Sequencing by Incorporation

ABSTRACT

Nucleotides and nucleotide analogs are used in various sequencing by incorporation/sequencing by synthesis methods. Nucleotide analogs comprising 3′-blocking groups are used to provide reversible chain-termination for sequencing by synthesis. Typical blocking groups include phosphate groups and carbamate groups. Fluorescent nucleotides are used to perform sequencing by synthesis with detection by incorporation of the fluorescently labeled nucleotide, optionally followed by photobleaching and intercalating dyes are used to detect addition of a non-labeled nucleotide in sequencing by synthesis with detection by intercalation. Microfluidic devices, including particle arrays, are used in the sequencing methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/413,049 filed Apr. 14,2003, which is a divisional of U.S. patent application Ser. No.09/510,205, filed Feb. 22, 2000, which claims benefit of and priority toU.S. Ser. No. 60/121,223, entitled “Manipulation of Microparticles inMicrofluidic Systems,” filed Feb. 23, 1999 by Mehta et al.; U.S. Ser.No. 60/127,825 entitled “Manipulation of Microparticles in MicrofluidicSystems,” filed Apr. 5, 1999 by Mehta et al.; U.S. Ser. No. 60/128,643entitled “Manipulation of Microparticles in Microfluidic Systems,” filedApr. 9, 1999 by Mehta et al.; co-filed U.S. application “Manipulation ofMicroparticles in Microfluidic Systems,” filed Feb. 22, 2000, AttorneyDocket No. 01-006830US by Mehta et al.; co-filed PCT applicationentitled “Manipulation of Microparticles in Microfluidic Systems,” filedFeb. 22, 2000, Attorney Docket No. 01-006830PC by Mehta et al.; andco-filed PCT application entitled “Sequencing by Incorporation” filedFeb. 22, 2000, Attorney Docket No. 01-006840PC by Parce et al.

BACKGROUND OF THE INVENTION

Most DNA sequencing today is carried out by chain termination methods ofDNA sequencing. The most popular chain termination methods of DNAsequencing are variants of the dideoxynucleotide mediated chaintermination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad.Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing,see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (e.g., Supplement 38, current through1998) (Ausubel), Chapter 7. Thousands of laboratories employdideoxynucleotide chain termination techniques. Commercial kitscontaining the reagents most typically used for these methods of DNAsequencing are available and widely used. In addition to the Sangermethods of chain termination, new PCR exonuclease digestion methods havealso been developed for DNA sequencing. Direct sequencing of PCRgenerated amplicons by selectively incorporating boronated nucleaseresistant nucleotides into the amplicons during PCR and digestion of theamplicons with a nuclease to produce sized template fragments has beenperformed (Porter et al. (1997) Nucleic Acids Research 25(8):1611-1617).The above methods typically require that the terminated fragments besequenced upon completion of the reaction. This is a time consuming stepthat limits the ability to sequence in a high throughput manner.

The development of microfluidic technologies by the inventors and theirco-workers has provided a fundamental shift in how artificial biologicaland chemical processes are performed. In particular, the inventors andtheir co-workers have provided microfluidic systems that dramaticallyincrease throughput for biological and chemical methods, as well asgreatly reducing reagent costs for the methods. In these microfluidicsystems, small volumes of fluid are moved through microchannels byelectrokinetic or pressure-based mechanisms. Fluids can be mixed, andthe results of the mixing experiments determined by monitoring adetectable signal from products of the mixing experiments.

Complete integrated systems with fluid handling, signal detection,sample storage and sample accessing are available. For example, Parce etal. “High Throughput Screening Assay Systems in Microscale FluidicDevices” WO 98/00231 and Knapp et al. “Closed Loop BiochemicalAnalyzers” (WO 98/45481; PCT/US98/06723) provide pioneering technologyfor the integration of microfluidics and sample selection andmanipulation. For example, in WO 98/45481, microfluidic apparatus,methods and integrated systems are provided for performing a largenumber of iterative, successive, or parallel fluid manipulations. Forexample, integrated sequencing systems, apparatus and methods areprovided for sequencing nucleic acids. This ability to iterativelysequence a large nucleic acid (or a large number of nucleic acids)provides for increased rates of sequencing, as well as lower sequencingreagent costs. Applications to compound screening, enzyme kineticdetermination, nucleic acid hybridization kinetics and many otherprocesses are also described by Knapp et al.

New or improved methods of sequencing are accordingly desirable,particularly those that take advantage of high-throughput, low costmicrofluidic systems. The present invention provides these and otherfeatures by providing new sequencing methods and high throughputmicroscale systems for providing sequencing reactions as well as manyother features that will be apparent upon complete review of thefollowing disclosure.

SUMMARY OF THE INVENTION

The present invention provides novel methods of sequencing by synthesisor incorporation. Nucleotides or nucleotide analogs are added toreaction mixtures comprising nucleic acid templates and primers, e.g.,DNA or RNA. The nucleotides are incorporated into the primer, resultingin an extended primer. The sequence is determined as each additionalcomplementary nucleotide is incorporated into the primer and the stepsare repeated until the entire template sequence or a portion thereof isdetermined.

In one embodiment, the nucleotides or nucleotide analogs, or a fractionthereof, comprise a 3′-blocking group and a detectable label moiety,which typically comprises a phosphate or a carbamate group. The3′-blocking groups provide reversible chain termination. When added to agrowing nucleic acid chain, these nucleotide analogs result in anon-extendable primer. The 3′-blocking group is typically removed, e.g.,by a reducing agent and/or a phosphatase, to produce an extendableprimer to which further nucleotides are added, thereby allowingcontinued sequencing of the nucleic acid template. Removal of the3′-blocking group is optionally performed before or after detection ofthe added nucleotide.

In another embodiment, the nucleotides or nucleotide analogs comprise afluorescent label. Sequencing by synthesis using fluorescent nucleotidestypically involves photobleaching the fluorescent label after detectingan added nucleotide. Photobleaching comprises applying a light pulsethat destroys or reduces to an acceptable level, e.g., a backgroundlevel or to a low enough level to prevent signal buildup over severalsequencing cycles, the fluorescence of the nucleotides, e.g., afluorescent nucleotide that has been added to the primer. The lightpulse is typically applied for about 20 seconds or less, about 10seconds or less, about 2 seconds or less, about 1 second or less, orabout 0.1 second or less. The light pulse typically has a wavelengthequal to the wavelength of light absorbed by the fluorescently labelednucleotides. Detection of the added fluorescently labeled nucleotideoccurs prior to or concurrent with photobleaching of the fluorescentlylabeled nucleotides and/or the extended primer. Nucleic acid templatescomprising about 50 or more nucleotides, about 100 or more nucleotides,about 500 or more nucleotides, about 1000 or more nucleotides, about2000 or more nucleotides, or about 10,000 or more nucleotides areoptionally sequenced using these methods, e.g., sequenced with at leastabout 80%, at least about 90%, or at least about 95% accuracy.

In another embodiment, sequencing comprises sequencing by synthesisusing detection of intercalating dyes (“sequencing by intercalation”).An intercalating dye is incubated or mixed with the template and primeras the sequencing reactions occur. When a nucleotide, e.g., a naturallyoccurring, non-labeled nucleotide, is incorporated into the primer, itforms an extended double-stranded region, into which intercalating dyesinsert, e.g., between the stacked bases. The intercalating dye isdetected, thus detecting the addition of a nucleotide to the growingchain and sequencing the template nucleic acid. The intercalating dyeoptionally comprises ethidium, ethidium bromide, an acridine dye, anintercalating nucleic acid stain, a cyanine dye, such as SYBR green,proflavin, propidium iodide, acriflavin, proflavin, actinomycin,anthracyclines, or nogalamycin. In some embodiments, photobleaching isperformed after detecting the intercalating dye or approximatelyconcurrent with detecting the intercalating dye.

The nucleotides or nucleotide analogs in the present invention typicallycomprise nucleoside 5′-triphosphates (dNTPs), e.g., deoxyadenosine5′-triphosphate (dATP), deoxyguanosine 5′-triphosphate (dGTP),deoxycytidine 5′-triphosphate (dCTP), deoxythymidine 5′-triphosphate(dTTP), deoxyuridine 5′-triphosphate (dUTP), adenosine 5′-triphosphate(ATP), guanosine 5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP),uridine 5′-triphosphate (UTP), or analogs thereof.

In some embodiments, the nucleotides or nucleotide analogs, or afraction thereof, comprise a detectable label moiety, e.g., afluorescent or chemiluminescent label moiety. Different nucleotidesoptionally comprise detectably different labels, e.g., ATP, GTP, CTP,TTP, and UTP each optionally comprising a distinguishable label.

The nucleotides are optionally incubated with the nucleic acids inseries or in combination. For example, four detectably differentnucleotides, e.g., reversible chain terminating nucleotide analogs, areoptionally simultaneously incubated with template, primer, andpolymerase. The added nucleotide stops chain growth, is detected, andthen the chain terminating portion of the nucleotide is removed, e.g.,the 3′-blocking group is removed to allow further extension of theprimer. For example, the 3′-blocking group is optionally removed in abuffer or wash that also removes all unincorporated nucleotides.Alternatively, the 3′-blocking group comprises the label moiety and isdetected after removal.

Alternatively, four nucleotides are added in series, one after theother. For example, one nucleotide is added, unincorporated nucleotidesare removed from the reaction, and a fluorescent signal is detected,e.g., from a fluorescently labeled nucleotide added to the primer chainor from an intercalating dye that has intercalated into the recentlyextended double-stranded nucleic acid region. A second nucleotide isadded, a third, and so on, e.g., until the nucleic acid template or aportion thereof is sequenced.

In another aspect, the methods involve performing the sequencing, e.g.,by incorporation, by photobleaching, by intercalation, and the like, ina microfluidic device. Nucleic acid templates, e.g., DNA or RNA, andprimers, are flowed through a microscale channel and contacted by apolymerase and one or more nucleotides or nucleotide analogs in themicroscale channel, thereby adding at least one of the one or morenucleotides or nucleotide analogs to the primer. The added nucleotide ornucleotide analog is detected and the steps are repeated, e.g., toobtain an entire sequence or a portion thereof.

In one embodiment, the template and/or primer are attached to a set ofparticles, e.g., an ordered array of particles, which is flowed througha microscale channel or positioned, e.g., in a fixed location, withinthe microscale channel. The sequencing reagents, e.g., a train ofreagents, are flowed across the particles to sequence the templatenucleic acid. Unincorporated nucleotides or reagents are flowed throughthe microchannel, e.g., to a waste reservoir. Alternatively, theparticle sets are flowed through the train of reagents to perform thesequencing. In some embodiments, the reagents are attached to particlesets and the template is flowed through the particle sets to besequenced. When a nucleotide or nucleotide analog is added to theprimer, a signal is typically detected from the added nucleotide, e.g.,on the particle sets or released from the particle set and flowedthrough a detection region.

The particle sets optionally comprise about 1 or more particles, about10 or more particles, about 100 or more particles, about 1000 or moreparticles, or about 10,000 or more particles. In some embodiments, theset of particles comprises a set of beads, which beads are selectedfrom: polymer beads, silica beads, ceramic beads, clay beads, glassbeads, magnetic beads, metallic beads, paramagnetic beads, inorganicbeads, and organic beads; and wherein the beads have a shape, whichshape is selected from one or more of: spherical, helical, cylindrical,spheroid, irregular, rod-shaped, cone-shaped, cubic, and polyhedral.

The train of reagents that is used to perform sequencing of a nucleicacid template typically comprises sequencing reagents for performingsequencing, e.g., sequencing by synthesis, e.g., with detection byphotobleaching, by pyrosequencing chemistry, or by intercalation.Typical reagents include, but are not limited to, one or more of: atemplate, a primer, a polymerase, a sufurylase, an apyrase, an inorganicphosphate, ATP, a thermostable polymerase, luciferin, luciferase, anendonuclease, an exonuclease, Mg⁺⁺ a molecular crowding agent, a buffer,a dNTP, a salt, a phosphatase, a reducing agent, a modified dNTP, anucleotide, a nucleotide analog, a nucleotide analog comprising a 3′-blocking group, a nucleotide analog comprising a 3 ′-phosphate group,a nucleotide analog comprising a 3′-carbamate group, a chain-terminatingnucleotide analog, a reversible chain terminating nucleotide analog, afluorescently labeled nucleotide, and an intercalating dye.

The particles and the reagent train are typically flowed through themicroscale channel by one or more of: pressure, centripetal force,centrifugal force, a moving magnetic field, and an electrokinetic force.

Microfluidic devices for sequencing a nucleic acid are also provided.The devices typically comprise a body structure having a microscalecavity disposed therein; and a set of particles, e.g., an ordered arrayof particles as described above, disposed within the microscale cavity.The set of particles comprises at least one set of nucleic acidtemplates and at least one set of nucleic acid primers. Nucleotidesand/or nucleotide analogs, as described above, are also disposed withinthe device, e.g., in reservoirs or attached to one or more particlesets.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic illustration of a particle set in a microscale channelor capillary useful for sequencing, e.g., a nucleic acid template, bysynthesis or incorporation.

FIG. 2: Panels A, B, and C are schematic drawings of an integratedsystem of the invention, including a body structure, microfabricatedelements, and a pipettor channel.

FIG. 3: Schematic drawing of an integrated system of the inventionfurther depicting incorporation of a microwell plate, a computer,detector and a fluid direction system. The integrated system isoptionally used with any suitable microfluidic device.

FIG. 4: Panels A, B, and C illustrate a DNA sequencing scheme usingphosphate/disulfide blocking groups.

FIG. 5: Side-view schematic of a main channel with reagent introductionchannels for sequencing nucleic acids

FIG. 6: Side view schematic of a capillary or microchannel comprising anintegral or formed porous barrier made from a set of particles. Theporous barrier is used, e.g., to capture multiple packets, i.e., sets ofparticles.

FIG. 7: Schematic illustration of sequencing by synthesis in a highthroughput system.

FIG. 8: Schematic of a microfluidic device useful in sequencing bysynthesis.

DETAILED DISCUSSION OF THE INVENTION

The present invention provides methods of sequencing nucleic acids bysynthesis or incorporation.

Chemical Structure Definitions

As used herein formula (I) refers to a compound having the formula:

wherein R⁴ comprises one or more of a linker moiety and a detectablelabel and B comprises one or more of a nitrogenous base and thedetectable label. A detectable label typically comprises fluorescent orchemiluminescent moiety, e.g., to detect the nucleotide after it hasbeen added to a growing primer strand. A “label” is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include fluorescent dyes (e.g., fluorescein, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P,³³P, etc.), enzymes (e.g., horse-radish peroxidaase, alkalinephosphatase etc.), and colorimetric labels such as gold colored glass orplastic e.g., polystyrene, polypropylene, latex, etc.) beads. Preferredlabel moieties in the present invention include, but are not limited to,fluorescein and rhodamine.

The label is coupled directly or indirectly to a component of the assayaccording to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation with the nucleotide,nucleoside, nitrogenous base, or the like, stability requirements,available instrumentation and disposal provisions. Non-radioactivelabels are often attached by indirect means. Generally, a ligandmolecule (e.g., biotin) is covalently bound to the component to belabeled. For example, a label is optionally covalently bound to thenitrogenous base moiety of a nucleotide or to the sugar moiety, e.g., atthe 3′-position, through a linker bound to the 3′position of anucleotide, or to a 3′-blocking group. The ligand then binds to ananti-ligand (e.g., streptavidin) molecule which is either inherentlydetectable or covalently bound to a signal system, such as a detectableenzyme, a fluorescent compound, or a chemiluminescent compound. A numberof ligands and anti-ligands are optionally used. Where a ligand has anatural anti-ligand, for example, biotin, thyroxine, or cortisol, it isused in conjunction with the labeled naturally occurring anti-ligand.Alternatively, any haptogenic or antigenic compound is used incombination with an antibody (see, e.g., Coligan (1991) CurrentProtocols in Immunology, Wiley/Greene, NY; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY for ageneral discussion of how to make and use antibodies). The components ofthe invention are also optionally conjugated directly tosignal-generating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include, e.g., luciferinand 2,3,-dihydrophthalalzinediones, e.g., luminol.

A linker moiety or linker molecule in the present invention typicallyconnects a 3′-blocking group to a detectable label. In some embodiments,the linker forms a portion of the 3′-blocking group. Linkers that areoptionally used to provide a detectable label moiety are describedabove. Other chemical linkers include, but are not limited to, an acyl,an S-acyl, an alkyl, an aromatic, an acetyl, or an heteroaromatic group,or the like.

A nitrogenous base typically comprises a heterocyclic base such asadenine, guanine, thymine, cytosine, or any other purines, pyrimidinesor derivative thereof.

Formula (II) refers to any compound having the formula:

wherein B comprises one or more of a nitrogenous base and the detectablelabel, as described above. Alternatively, a label moiety is attached tothe carbamate linker group.

Formula (III) refers to any compound having the formula:

wherein R¹ comprises a nucleoside, a nucleotide, a nucleotide analog, anucleoside analog, or the 3′-end of a growing nucleic acid chain, e.g.,a primer, R² comprises a blocking moiety, which blocking moietycomprises a detectable label, and R³ comprises a hydrogen or a negativecharge. The blocking moiety comprises any chemical or biological moietythat prevents addition of another nucleotide or nucleotide analog to thegrowing nucleic acid, e.g., to R¹, and is removable, e.g., chemically orenzymatically. Removal of the blocking group typically results in amolecule having formula (VII) or formula (VIII) as described below.

Formula (IV) refers to any compound having the formula:

wherein B comprises a nitrogenous base, as described above.

Formula (V) refers to any compound having the formula:

wherein R⁴ comprises one or more of: a linker moiety and a detectablelabel. Formula (V) optionally serves as the blocking moiety for formula(III).

Formula (VI) refers to any compound having the formula:

wherein R⁴ comprises one or more of: a linker moiety and a detectablelabel. Formula (VI) also provides an example blocking group as used informula (III).

Formula (VII) refers to any compound having the formula:

wherein R¹ comprises a nucleotide, nucleotide analog, nucleoside,nucleoside analog, a nucleic acid, a primer, or the like.

Formula (VIII) refers to any compound having the formula:

wherein R¹ comprises a nucleotide, nucleoside, nucleotide analog ornucleoside analog, a nucleic acid, a primer, or the like.

Formula (IX) refers to any compound having the formula:

wherein R¹ comprises a nucleoside, a nucleotide, a nucleoside analog, anucleotide analog, a nucleic acid, a primer, or the like and R²comprises a linker moiety, and either R¹ or R² further comprises adetectable label. For example, a detectable label is optionally attachedto the nitrogenous base of R¹.

Formula (X) refers to any compound having the formula:

Formula (X) provides an example linker molecule (R²) for formula (IX).I. Introduction

The present invention provides novel methods for sequencing nucleicacids, e.g., in microfluidic devices, e.g., using particle arrays. Thesequencing methods provided comprise sequencing by incorporation orsynthesis. Sequencing by incorporation refers to a method of determiningthe sequence or order of nucleotides in a nucleic acid, e.g., DNA orRNA, e.g., without chain degradation or termination and subsequentseparation. A nucleotide or nucleotide analog is added to a primerstrand, e.g., complementary to the template strand, and detected asadded. Additional nucleotides are then added to the same primer strands,i.e., the strands are not permanently terminated. In some embodiments,the growing primer strand is reversibly terminated, i.e., it istemporarily terminated and then termination is reversed, e.g., byremoval of a blocking group. In other embodiments, a fraction of thechains are terminated while another fraction is synthesized to the end,with detection after each nucleotide addition.

The present invention provides at least four new methods of sequencingby synthesis: (1) sequencing using a 3′-phosphate blocking group; (2)sequencing using a 3′-carbamate blocking group; (3) sequencing bysynthesis with detection by photobleaching (“sequencing byphotobleaching”); and (4) sequencing by synthesis using detection ofintercalating dyes (“sequencing by intercalation”).

Sequencing using blocking groups, e.g., phosphate and carbamatenucleotide analogs, typically involves reversibly terminating growingnucleic acid strands. For example, the presence of the blocking groupprevents additional nucleotides from being incorporated into the primerstrand, but the blocking group is removable or cleavable allowingsynthesis of the primer strand to continue when desired, e.g., afterdetection.

Sequencing by photobleaching typically involves the uses offluorescently labeled nucleotides to synthesize a primer nucleic acidthat is complementary to the template nucleic acid. Photobleachingreduces the intensity of the signal, which builds with each addition ofa fluorescently labeled nucleotide to the primer strand. By reducing thesignal intensity, longer DNA templates are optionally sequenced.

Sequencing by intercalation relies on an intercalating dye to providedetection of an added nucleotide. Nucleotides are added one at a time toa growing strand and detected due to a signal from an intercalating dyethat is differentially associated with the extended nucleic acid strand.The dye inserts between the stacked bases of a double helical nucleicacid. As the primer strand grows, the double-stranded region continuallyincreases in length, e.g., until it is as long as the template or adesired length. The more bases that are added to the primer, the moreintercalation occurs, thus providing a signal increase from which theaddition of a nucleotide is detected. In some embodiments, theintercalating dyes are photobleached after incorporation to reducesignal intensity.

The above methods all represent types of sequencing by synthesis becauseat least a portion of the growing primer strands are synthesized, e.g.,to the end, as opposed to being terminated, e.g., mid-length. Themethods are optionally practiced in microfluidic devices, e.g., usingparticle arrays, in capillaries, in microwell plates, or the like.

II. General Description of Sequencing by Incorporation

The present invention provides a plurality of methods for sequencing bysynthesis or incorporation. In particular, reversible chain terminationmethods are provided. For example, primer strands are terminated by theaddition of a nucleotide comprising a blocking group and then theblocking group is removed to allow further elongation. In a secondembodiment, sequencing by synthesis is performed using fluorescentlylabeled nucleotides and periodically photobleaching the growing primerstrand to reduce fluorescent signal build up. Alternatively, the signalis allowed to build up and detected without photobleaching. In a thirdembodiment, unlabeled nucleotides are added to growing primer strandsand detected by detecting an increase in intercalation. Intercalationincreases as the length of the double strand nucleic acid increases.Therefore the signal level increases as each additional complementarynucleotide is added to the growing primer/template strand.

A number of basic sequencing by incorporation methods are known, e.g.,as set forth in Hyman U.S. Pat. No. 4,971,903; Malemede U.S. Pat. No.4,863,849; Cheeseman U.S. Pat. No. 5,302,509, and Canard U.S. Pat. No.5,798,210. Generally, any detectable event associated with incorporationof a nucleotide can be used to monitor sequencing reactions. Insequencing by incorporation methods, incorporation of nucleotides ornucleotide analogs into nucleic acids, e.g., using a polymerase toextend a primer hybridized to a complementary template nucleic acid, ismonitored to provide an indication of the sequence of the templatenucleic acid. This can be performed by selectively adding reagentscomprising labels such as bases comprising fluorescent moieties, e.g.,four detectably different fluorescent moieties, to e.g., a member of anarray set comprising the template nucleic acid and monitoringincorporation of the label into the nucleic acid. The present inventionprovides new or improved methods of sequencing by incorporation, e.g.,by using alternative detectable events, such as the addition of anintercalator to a double-helix; the addition of a labeled nucleotide toa primer, by providing reversible chain terminating nucleotides; or byphotobleaching to insure detectable addition of nucleotides at anydesired read length.

Generally, sequencing by incorporation involves providing a nucleic acidtemplate and a primer, which are hybridized to form a double-strandednucleic acid region, which is sequentially extended by addition ofcomplementary nucleotides to the primer strand. “Nucleic acid template”refers to a polynucleotide chain utilized, e.g., during DNA replicationor transcription, as a guide to the synthesis of a second polynucleotidechain with a complementary base sequence. The template nucleic acids ofthe present invention typically comprise DNA or RNA and typicallycomprises about 50 or more, about 500 or more, about 1000 or more, about2000 or more nucleotides, or about 10,000 or more nucleotides.

The template is typically hybridized or annealed to a primer, forming adouble stranded nucleic acid region that is extended by adding bases tothe primer strand. The primer comprises a short stretch of nucleotides,e.g., DNA or RNA, that is elongated by a polymerase, e.g., a DNApolymerase, taq polymerase, other thermostable polymerases, roomtemperature polymerases, and the like. The elongated or extended primeris synthesized in the presence of polymerase and one or more nucleotidesor nucleotide analogs to produce a nucleic acid that is complementary tothe template strand. Each addition of a nucleotide or nucleotide analogextends the double-stranded region of the nucleic acid.

The nucleic acid and primer are typically hybridized and incubated ormixed with one or more nucleotides or nucleotide analogs in the presenceof a polymerase. The nucleotides are added to the primer strand toproduce an extended primer. The extended primer is a nucleic acid primerthat has had one or more nucleotides added to it, e.g., nucleotides thatare complementary to the annealed template. Each added nucleotide isdetected to determine the sequence of the template. The term,“nucleotides” is used herein to refer to the building blocks of nucleicacids, including, but not limited to, naturally occurring andnon-naturally occurring nucleotides, nucleosides, nucleotide analogs,nucleoside analogs, and the like. Nucleosides typically comprise anitrogenous base and a sugar, e.g., deoxyribose, ribose, or the like.Nucleotides generally comprise a nitrogenous base, a sugar, and aphosphate group, e.g., a monophosphate, a diphosphate, or atriphosphate. “Nitrogenous base” refers to heterocyclic bases such asadenine, guanine, thymine, cytosine, other purines and pyrimidines andderivatives thereof. Nucleotides used in the present invention typicallycomprise 5′-nucleoside phosphates including, but not limited to,deoxyadenosine 5′-triphosphate, deoxyguanosine 5′-triphosphate,deoxycytidine 5′-triphosphate, deoxythymidine 5′-triphosphate,deoxyuridine 5′-triphosphate, adenosine 5′-triphosphate, guanosine5′-triphosphate, cytidine 5′-triphosphate, uridine 5′-triphosphate,thymidine-5′-triphosphate, and analogs thereof.

“Nucleotide analog” is used herein to refer to compounds, e.g.,derivatives of nucleosides and nucleotides, that are optionallyincorporated into a growing nucleic acid chain, e.g., added to a primeror extended primer to form an extended double stranded region. Preferrednucleotide analogs include, but are not limited to, compounds comprisinga 3′-blocking group, e.g., a chain terminating blocking group or areversible chain terminating blocking group. Blocking groups of thepresent invention typically comprises a phosphate or a carbamate group.Preferred nucleotide analogs include, but are not limited to, compoundscomprising one or more of the structures represented by formulas (I),(II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), as describedabove.

Other nucleotide analogs of the present invention include nucleotidescomprising a label moiety, e.g., a detectable label moiety. Detectablelabel moieties include, but are not limited to, fluorescent moieties andchemiluminescent moieties. Nucleotide and nucleoside analogs of thepresent invention are optionally synthetic, naturally occurring, ornon-naturally occurring compounds that have similar properties tonucleotides and nucleosides and are typically metabolized in a similarmanner. Other examples include, but are not limited to,phosphorothioates, phosphoroamidates, methyl phosphonates,chiral-methylphosphonates, 2-O-methyl ribonucleotides,dideoxynucleotides, boronated nucleotides, and the like.

When chain terminating nucleotide analogs are used to extend a primer ina sequencing reaction, the extended primer becomes a non-extendableprimer upon addition of the chain terminating nucleotide analog. A“non-extendable primer” is a primer or nucleic acid fragment to whichthe relevant polymerase, i.e., the polymerase in the reaction mixture,will not add another nucleotide or nucleotide analog, i.e., because the3′-OH group is blocked, e.g., by a carbamate or phosphate group. Thechain terminates and no more nucleotides are added.

In the present invention, a preferred nucleotide analog is a reversiblechain terminating nucleotide. These nucleotides, when added to a growingprimer chain, terminate the chain by the addition of the nucleotideanalog, e.g., the blocking group, and thereby inhibit the addition ofany other nucleotides. The terminated primer is then subjected to areaction that reverses the termination. For example, adding a nucleotidecomprising a 3′-blocking group to a growing nucleic acid chainterminates the chain and inhibits further additions of nucleotides.However, the chain terminating 3′-blocking group is optionally removedto allow addition or growth of the chain to continue. For example,reagents that destroy the blocking group are optionally added to thereaction mixtures. Preferred reversible chain terminating nucleotidesand blocking groups are described in more detail below. Non-terminatingnucleotides and nucleotide analogs are those that allow further additionof nucleotides to a growing chain of nucleotides. For example, anon-terminating nucleotide typically contains a 3′-OH group so thatanother nucleotide is optionally added to the 3′-terminus of the growingnucleic acid chain. The chain terminating nucleotides typically containa blocking group on the 3′OH. A “blocking group” typically preventsaddition of a nucleotide to the 3′-terminus of a nucleic acid. Blockinggroups are typically chemical moieties that are attached to the nucleicacid or nucleotide in the 3′-position to prevent further binding orreactions at that position. Preferred blocking groups of the presentinvention include, but are not limited to, phosphate and carbamategroups, e.g., 3′-phosphates and 3′-carbamates. For more information onblocking groups, see, e.g., Protective Groups in Organic Synthesis, byT. Greene, Wiley and Sons, New York (1981).

After adding a nucleotide, e.g., to a growing primer chain,unincorporated nucleotides are optionally removed from the reaction,e.g., by washing a channel or capillary, e.g., with a buffer. Addednucleotides are then detected, e.g., by detecting a fluorescent orchemiluminescent signal from the added nucleotide. “Unincorporatednucleotides” are those nucleotides that were not incorporated or addedinto the nucleic acid chain. In some embodiments, the unincorporatednucleotides are left in the reaction but rendered unincorporable, e.g.,by chemical reaction or change in reaction conditions.

The present invention provides a plurality of methods for sequencing bysynthesis, e.g., sequencing using 3′-blocking groups such as phosphategroups and carbamates groups, sequencing by photobleaching, sequencingusing a low concentration of chain terminating labeled nucleotides incombination with non-terminating, non-labeled nucleotides, sequencing bysynthesis using detection by intercalation, and the like. These methodsare each discussed in more detail below

In a preferred method, the sequencing reactions of the present inventionare performed using immobilized templates and primers. For example, thetemplates and primers are optionally immobilized on a membrane or porousmatrix or on the walls of a capillary, microfluidic channel ormicrowell. Alternatively, the template and primer are immobilized on aset of particles, which particles are then optionally flowed through acapillary or channels/chambers of a microfluidic device.

For example, the template and the primer are optionally attached to aparticle array. The particle array is flowed through or positionedwithin, e.g., a capillary or microfluidic device. Sequencing reagentsare optionally flowed across the particle array to sequence the templateor the particle array is flowed through a train of reagents. Particlearrays are discussed in more detail in U.S. Ser. No. 60/128,643, filedApr. 9, 1999 and in co-filed application, “Manipulation ofMicroparticles in Microfluidic Systems,” by Mehta et al., AttorneyDocket No. 01-068-30, which is hereby incorporated by reference.

When performed in a microfluidic device, the methods presented hereintypically comprise flowing a nucleic acid template and a primer througha microscale channel, in which they are typically immobilized,hybridized and sequenced. Alternatively, a microfluidic devicecomprising a template and primer disposed therein is provided.Sequencing reagents, e.g., polymerase solutions, nucleotides, buffersand the like, are optionally flowed across the template and primer orthe template and primer are flowed through the reagents. When thenucleotides and polymerase are incubated with the template and theprimer, e.g., by flowing the nucleotides and polymerase across thetemplate and primer, one or more nucleotides, e.g., complementarynucleotides, are optionally incorporated into the primer, producing anextended primer. The channel is optionally washed to remove anyunincorporated nucleotides and then the added nucleotide is detected.The steps are then repeated to provide, e.g., the entire sequence of thetemplate nucleic acid or a portion thereof.

In addition to providing novel sequencing methods, the present inventionprovides microfluidic sequencing methods. Sequencing by incorporationusing any method described herein is optionally performed in amicrofluidic device. In addition conventional sequencing methods areoptionally improved by the use of microfluidic devices.

It is expected that one of skill is familiar with fundamental sequencingmethodologies applicable to the present invention. Examples oftechniques for making and sequencing nucleic acids by conventionalmethods, and instructions sufficient to direct persons of skill throughmost standard cloning and other template preparation exercises are foundin Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.)Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,(Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1997, supplement 37)(Ausubel). Basic procedures for cloning and other aspects of molecularbiology and underlying theoretical considerations are also found inLewin (1995) Genes V Oxford University Press Inc., NY (Lewin); andWatson et al. (1992) Recombinant DNA Second Edition Scientific AmericanBooks, NY. Product information from manufacturers of biological reagentsand experimental equipment also provide information useful in knownbiological methods. Such manufacturers include the Sigma ChemicalCompany (Saint Louis, Mo.); New England Biolabs (Beverly, Mass.); R&Dsystems (Minneapolis, Minn.); Pharmacia LKB Biotechnology (Piscataway,N.J.); CLONTECH Laboratories, Inc. (Palo Alto, Calif.); ChemGenes Corp.,(Waltham Mass.) Aldrich Chemical Company (Milwaukee, Wis.); GlenResearch, Inc. (Sterling, Va.); GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.); Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland); Invitrogen (San Diego, Calif.); Perkin Elmer(Foster City, Calif.); and Strategene; as well as many other commercialsources known to one of skill.

In one aspect, the generation of large nucleic acids is useful inpracticing the invention, e.g., as templates fixed to array members,e.g., for sequencing long regions of nucleic acids, or for monitoringexpression products by hybridization of biological materials to thefixed templates. It will be appreciated that such templates areparticularly useful in some aspects where the methods and devices of theinvention are used to sequence large regions of DNA, e.g., for genomicstypes of applications. An introduction to large clones such as YACs,BACs, PACs and MACs as artificial chromosomes is provided by Monaco andLarin (1994) Trends Biotechnol 12 (7): 280-286.

The construction of nucleic acid libraries of template nucleic acids isdescribed in the above references. YACs and YAC libraries are furtherdescribed in Burke et al. (1987) Science 236:806-812. Gridded librariesof YACs are described in Anand et al. (1989) Nucleic Acids Res. 17,3425-3433, and Anand et al. (1990) Nucleic Acids Res. Riley (1990)18:1951-1956 Nucleic Acids Res. 18(10): 2887-2890 and the referencestherein describe cloning of YACs and the use of vectorettes inconjunction with YACs. See also, Ausubel, chapter 13. Cosmid cloning isalso well known. See, e.g., Ausubel, chapter 1.10.11 (supplement 13) andthe references therein. See also, Ish-Horowitz and Burke (1981) NucleicAcids Res. 9:2989-2998; Murray (1983) Phage Lambda and Molecular Cloningin Lambda II (Hendrix et al., eds) 395-432 Cold Spring HarborLaboratory, NY; Frischauf et al. (1983) J. Mol. Biol. 170:827-842; and,Dunn and Blattner (1987) Nucleic Acids Res. 15:2677-2698, and thereferences cited therein. Construction of BAC and P1 libraries is wellknown; see, e.g., Ashworth et al. (1995) Anal Biochem 224 (2): 564-571;Wang et al. (1994) Genomics 24(3): 527-534; Kim et al. (1994) Genomics22(2): 336-9; Rouquier et al. (1994) Anal Biochem 217(2): 205-9; Shizuyaet al. (1992) Proc Natl Acad Sci U S A 89(18): 8794-7; Kim et al. (1994)Genomics 22 (2): 336-9; Woo et al. (1994) Nucleic Acids Res 22(23):4922-31; Wang et al. (1995) Plant (3): 525-33; Cai (1995) Genomics 29(2): 413-25; Schmitt et al. (1996) Genome Res 1996 33(1): 9-20; Kim etal. (1996) Genomics 34(2): 213-8; Kim et al. (1996) Proc Natl Acad Sci US A (13): 6297-301; Pusch et al. (1996) Gene 183(1-2): 29-33; and, Wanget al. (1996) Genome Res 6(7): 612-9.

In general, where the desired goal of a sequencing project is thesequencing of a genome or expression profile of an organism, a libraryof the organism's cDNA or genomic DNA is made according to standardprocedures described, e.g., in the references above. Individual clonesare isolated and sequenced, and overlapping sequence information isordered to provide the sequence of the organism. See also, Tomb et al.(1997) Nature 539-547 describing the whole genome random sequencing andassembly of the complete genomic sequence of Helicobacter pylori;Fleischmann et al. (1995) Science 269:496-512 describing whole genomerandom sequencing and assembly of the complete Haemophilus influenzaegenome; Fraser et al. (1995) Science 270:397-403 describing whole genomerandom sequencing and assembly of the complete Mycoplasma genitaliumgenome and Bult et al. (1996) Science 273:1058-1073 describing wholegenome random sequencing and assembly of the complete Methanococcusjannaschii genome.

III. Sequencing a Nucleic Acid Using Reversible Chain TerminatingNucleotide Analogs Comprising a 3′-Blocking Group

In one embodiment, the present invention provides methods of sequencingby synthesis using nucleotide analogs with reversible chain terminating3′-blocking groups. The blocking groups are used to temporarily stopnucleic acid synthesis after the addition of a nucleotide. Whilesynthesis is temporarily blocked or terminated, the newly incorporatednucleotide is detected. The blocking groups are then removed and chainelongation continues, e.g., until the entire template is sequenced.

A template and a primer are provided and hybridized, i.e., and typicallyimmobilized. For example, template and primer nucleic acids areoptionally flowed through a microfluidic device or immobilized in amicrowell plate. The template and primer are incubated with a polymeraseand one or more nucleotide analogs comprising reversible chainterminating blocking groups. Complementary nucleotides are added to theprimer strand, resulting in an extended primer, and detected, therebyproviding an indication of the template strand sequence based on theidentity of the nucleotide analog added to the primer. For examplerelevant nucleotides, e.g., C, A, G, T, and U, are optionallysimultaneously incubated with the template and primer or each relevantnucleotide is added separately. When added together, each type ofnucleotide comprises a detectably different label and detectionidentifies which nucleotide was added. When each of the nucleotides isadded individually, e.g., in series, the nucleotides optionally comprisethe same are different labels. In this case, detection determines if anucleotide was added and if not, then the next nucleotide in the seriesis added. The steps are repeated until the entire template or a portionthereof is sequenced.

With chain terminating nucleotides analogs, the addition of thenucleotide inhibits further elongation of the primer. In previouslyknown methods, some nucleic acids chains are terminated and sequencingcontinues with others and then all resulting fragments are separatedafter the sequencing reactions are complete and detected. In the presentmethods no separation is necessary, because the chain termination isreversible. The chain terminating group is removed from the growingnucleic acid chain after it is detected (or the group is optionallyremoved and then detected). The removal leaves an extendable primer towhich another nucleotide is added.

The chain terminating nucleotides used in the present inventiontypically comprise a phosphate group or a carbamate group, e.g., as informula (I), (II), (III) or (IX).

Phosphate Blocking Groups

In one embodiment, the blocking group of the present invention comprisesa phosphate moiety. When added to a growing nucleic acid chain, e.g., aprimer, the blocking group blocks nucleic aid growth until it isremoved. The blocking group typically comprises a phosphate moiety and alinker moiety. The linker moiety, which optionally comprises adetectable label, is typically removed by chemical cleavage and thephosphate is typically removed by enzymatic cleavage.

The phosphate blocking group of the invention is typically bound to the3′-OH position of a nucleotide, nucleotide analog, or nucleic acid. Thephosphate blocking groups of the present invention typically compriseone or more phosphate moieties, and a blocking moiety. The blockingmoiety typically comprises one or more of: a linker moiety, a disulfidemoiety, a compound having formula (V), a compound having formula (VI),and a detectable label. Linkers and labels are described above.Typically a linker moiety is any molecule or portion thereof used toattach a label to a nucleotide analog, e.g., in this case to attach thelabel moiety to a blocking moiety, such as that of formula (V) or (VI).Typical linker groups comprise alkyl, acyl, acetyl and aromaticmolecules. The most basic structure for a phosphate nucleotide analog ofthe invention is provided by Formula (III).

Nucleotides of interest are typically labeled, e.g., by attaching adetectable label to the 3′-blocking group. If all four nucleotides arelabeled with a different label, they are optionally incubated with thetemplate and primer in one batch and detection identifies whichnucleotide was added. If the same label is used for all nucleotides ofinterest, the nucleotides are incubated with the template primer inseries, detecting after each nucleotide, e.g., A, C, G, U, and T, in theseries.

Examples of nucleotide analogs having phosphate blocking groups include,but are not limited to compounds having formula (I), (III), (VII),(VIII), and the like. The phosphate groups are typically removed afterthey are detected, e.g., a nucleotide analog is added to the growingchain, detected, and removed. Typically, detection comprises detecting afluorescent moiety on the added nucleotide, e.g., to determine thenature or identity of the added nucleotide. A variety of fluorescentlabels and methods of attachment to nucleotides are well known to thoseof skill in the art. Similar phosphate groups have been generated foruse with protein derivatives. See, e.g., C. Mathe et al. J. Org. Chem.63, 8547-8550 (1998); Gay et al., J. Chem. Soc. 8, 1123 (1970); and,Eckstein at al. Trendes in Biochem 14, 97 (1989).

Removal of the phosphate blocking group comprises removal of the linkerand/or label moiety, removal of the blocking group moiety, and removalof the phosphate moiety. Removal of each section of the blocking groupis optionally a separate step or one step, e.g., removal of the blockingmoiety, the phosphate moiety and the linker moiety all occursimultaneously. For example, the linker and label moiety, e.g., afluorescent dye are typically removed using a reducing agent, e.g.,diborane, TCEP, disulfide reductase, dithiothreitol (DTT), glutathione,or the like. For further information reductions, e.g., of disulfides,see, e.g., March, Advanced Organic Chemistry, Fourth Edition, Wiley &Sons, New York, (1992). Removal of the linker and/or label typicallyresults in a compound of formula (VII), which self cleaves, e.g.,through an intramolecular nucleophilic attack, e.g., simultaneously withor right after the reduction, to produce a free 3′-phosphate group asshown in Formula (VIII). Any combination of linker moieties and blockinggroups that result in a compound of formula (VII) or (VIII) areoptionally used. For example, any arrangement of linkers, blockinggroups, dyes, and the like, which when reduced or otherwise cleavedproduces a compound of formula (VII), is optionally used to provide anucleotide analog of the present invention. Compound (VII) comprises anunstable compound. Having a thiol group at a specific distance, e.g.,the distance required for 2 CH₂ groups, from the phosphate moiety allowsthe compound to spontaneously degrade, e.g., through an intramolecularnucleophilic attack, e.g., sulfur acts as a nucleophile and causes theelimination of the blocking group from the phosphate group, to thecompound of formula (VIII).

The phosphate group is optionally removed with a phosphatase, e.g., analkaline phosphatase, a 3′-phosphatase, or any naturally occurring ornon-naturally occurring enzyme comprising 3′-phosphatase activity, e.g.,a kinase, e.g., T4 kinase, with 3′-alkaline phosphatase activity. Thephosphatase removes the phosphate group from the nucleotide ornucleotide analog of formula (VIII), resulting in a free 3′-OH which isoptionally extended by the addition of another nucleotide. Preferably,the phosphatase or other enzyme with phosphatase activity does notdegrade the phosphodiester bonds of the DNA backbone. Alternatively, the3′-phosphate is chemically cleaved.

For example, DTT and alkaline phosphatase are added to a sequencingreaction mixture, e.g., after detection of the most recently addednucleotide, to remove the blocking moiety, phosphate, linker and labelmoieties simultaneously. FIG. 4 provides a sequencing scheme using aphosphate blocking group that is removed as described above. One or morenucleotide analogs, e.g., with the bases: A, C, G, U, and T, areprovided (FIG. 4, Panel A) wherein each nucleotide comprise a3′-blocking group. The 3′-blocking group comprises a 3-′phosphate and adisulfide blocking group. In addition, the blocking group comprises anyappropriate linker that is used to attach a detectable label moiety tothe disulfide group. For example, A, C, G, and T are each given adifferent detectable label, e.g., four different fluorescent dyes.Methods of attaching labels, e.g., fluorescent dyes, to nucleotides andnucleotides analogs are well known to those of skill in the art. FIG. 4,Panel B illustrates the addition, e.g., in the presence of DNApolymerase, of the nucleotide from Panel A to the 3′-end of a growingDNA chain, e.g., a primer that is hybridized to the nucleic acidtemplate being sequenced. The nature of the nucleotide added, e.g., A,C, G, or T, is determined by the template which is being sequenced. Acomplementary nucleotide is added. The DNA chain is thereby extended byone nucleotide, e.g., a nucleotide that is complementary to the templatenucleic acid. However, the 3′-end comprises a blocking group thatprevents further extension because the 3′OH is not free.

Unincorporated nucleotides are then optionally removed from the reactionmixture. For example, the template and primer are optionally attached toa membrane and the unincorporated nucleotides are washed from themembrane. The added nucleotide is then optionally detected, its identitydetermined by the fluorescent signal detected. Alternatively, thetemplate is incubated with one base at a time, e.g., A, C, G, or U, ismixed in series with template until the complementary one is added anddetected.

The blocking group is then removed as shown in Panel C. The extended DNAchain is treated, e.g., with DTT, and alkaline phosphatase, resulting ina DNA chain with an extendable 3′OH. If not detected after removal ofunincorporated nucleotides, the blocking group comprising the label isoptionally detected after the removal of the blocking group from theextended DNA chain. The removed blocking groups provide a signal, e.g.,a fluorescent signal that is optionally detected, e.g., beforediscarding the removed blocking groups. The process is then repeated todetermine additional nucleotides in the sequence of the DNA template.

For further information on types of reducing agents and linkers that areoptionally cleaved, e.g., to produce a compound of formula (VII) or(VIII), see, e.g., March, Advanced Organic Chemistry, Fourth Edition,Wiley & Sons, New York, (1992); and Carey and Sundberg, Advanced OrganicChemistry, Parts I and II, Third Edition, Plenum Press, New York,(1990).

Carbamate-Blocking Groups

In other embodiments, nucleic acid synthesis is temporarily terminatedby a nucleotide comprising a removable carbamate-blocking group. Theblocking group comprises a carbamate linkage, e.g., between a linkerand/or label moiety and the 3′-position of a nucleic acid or nucleotide.The carbamate group is typically attached to the nucleotide ornucleotide analog such that it blocks the 3′-OH and thus blocksincorporation of additional nucleotides or nucleotide analogs, e.g., tothe growing primer strand. The carbamate group is optionally cleaved toallow further strand elongation and further sequencing.

A carbamate group is optionally added to a 3′-OH of a nucleotide ornucleic acid, e.g., through reactions with various amines. The carbamatelinkage is subject to attack, e.g., with an esterase, a mild base, or ahydroxyl amine, to produce a free 3′OH group. Therefore, the carbamatenucleotide analogs are optionally used as the analogs described above.The nucleotides are incubated with the template and primer and when anucleotide is added to the primer, it terminates the chain. However, thecarbamate linkage is then optionally cleaved to provide an extendableprimer.

Example carbamate nucleotides include, but are not limited to, those offormula (II) and (IX). The carbamate nucleotides are typically labeledand detected as described above. In some embodiments, the carbamate,which includes a detectable label is cleaved from the extended primerand then detected.

In one embodiment, the carbamate nucleotides analogs are used tosequence a nucleic acid in a capillary comprising, e.g., a particleretention element or particle capture region. For example FIG. 1provides capillary 105 and particle retention element 110. As shownparticle retention element comprises a set of particles, e.g., epoxycoated particles, which are immobilized in capillary 105. Particle set110 forms a particle capture region by forming a porous barrier. Inother embodiments, the porous barrier or particle retention element isoptionally a frit or a constriction in the channel, e.g., a narrowchannel region. Templates and/or primers are optionally attached toparticle set 115, e.g., through biotin-avidin binding orbiotin-streptavidin binding, either before or after the particle set hasbeen fixed in capillary 105. Particle set 115 is captured or retained byparticle set 110 because the mean diameter of the particles in particleset 115 is larger than the pore size created by particle set 110.Alternatively, particle set 115 is held in place by magnetic force or bychemically binding to the channel or capillary. A train of reagents isthen flowed across particle set 115 to sequence the template. Forexample a set of nucleotide analogs comprising carbamate blocking groupsis flowed across the template, thus adding a complementary base to theprimer. For example, nucleotide analogs comprising, e.g., ATP, GTP, CTP,and TTP with carbamate blocking groups and detectably different labelsare optionally flowed across particle set 115. Unincorporatednucleotides are removed from capillary 105, e.g., by washing a bufferthrough capillary 105, which buffer flows through porous barrier 110. Adetector proximal to particle set 115 is used to detect the addednucleotide, thus detecting the identity and sequencing the template. Thesteps are repeated, with each cycle identifying at least one nucleotidein the template sequence.

IV. Sequencing by Photobleaching

Typically, in sequencing by synthesis a fluorescently labeled nucleotideis detected as it is added to a growing nucleic acid chain, e.g., aprimer that is hybridized to a template. As more fluorescently labelednucleotides are added to the primer strand, the signal level increasesand the ability to detect the nucleotide addition decreases. In thepresent method, fluorescently labeled nucleotides are photobleachedafter incorporation to reduce the signal level and increase the templatenucleic acid read length.

The template is incubated with each different nucleotide in series andas a nucleotide is added to the primer, a signal is detected. Forexample, a nucleic acid template and primer are anchored or immobilized,e.g., on a membrane or on a capillary or microchannel wall, e.g.,through streptavidin-biotin binding. A polymerase and a fluorescentnucleotide or a mixture of fluorescent nucleotides and non-fluorescentnucleotides, e.g., A, G, C, or T, are incubated with the template andprimer, e.g., by flowing the nucleotides and polymerase across theimmobilized templates or by contacting a membrane with the polymeraseand nucleotides. Any of the nucleotides or nucleotide analogs in thepresent invention are optionally used. Fluorescently labelednucleotides, e.g., nucleoside-5′-triphosphates with a fluorescent labelmoiety attached, e.g., to the base, are preferred. If the labelednucleotide is complementary to the template, it is incorporated into thegrowing primer. Typically unincorporated nucleotides are removed beforedetection, e.g., by flowing buffer through the channel or across themembrane. A fluorescent signal is then detected from the incorporatednucleotides or nucleotide analogs. If a nucleotide is not incorporated,a signal is not detected, and the process is repeated with a differentnucleotide until the complementary nucleotide is determined.Alternatively, all four nucleotides are added together, e.g., when eachnucleotide is labeled with a detectably different fluorescent label.

Each time a fluorescent nucleotide is added to the growing chain, theoverall or background level of fluorescence increases, thereby making itmore difficult to detect a signal from a newly incorporated nucleotide.For example, to reduce the level of fluorescence and prevent previouslyincorporated nucleotides from interfering with the signals obtained, thesignals are photobleached.

Photobleaching comprises applying a light pulse to the nucleic acidprimer into which a fluorescent nucleotide has been incorporated. Thelight pulse typically comprises a wavelength equal to the wavelength oflight absorbed by the fluorescent nucleotide of interest. The pulse isapplied for about 50 seconds or less, about 20 seconds or less, about 10seconds or less, about 5 seconds or less, about 2 seconds or less, about1 seconds or less, or about 0. 1 second or less. The pulse destroys thefluorescence of the fluorescently labeled nucleotides and/or thefluorescently labeled primer or nucleic acid or reduces it to anacceptable level, e.g., a background level or a level that is low enoughto prevent signal buildup over several cycles. Background level istypically a signal level over which an additional fluorescent signal dueto the incorporation of an additional nucleotide to a growing nucleicacid chain is detectable. The fluorescence does not have to becompletely bleached out. In fact, the photobleach pulse is optionallyapplied for a photobleach half-life, i.e., the time it takes to reducethe fluorescence by one half. If a half-life photobleach time is used,the signal from the first nucleotides to be incorporated will eventuallybe reduced to background or substantially zero because each subsequentphotobleach pulse reduces the remaining fluorescence for anotherhalf-life each time a nucleotide is incorporate and photobleached. Forexample, if the pulse is applied for the bleach time half-life, one halfof the fluorescent intensity is bleached out. After three nucleotides orbases are added to the primer, the first fluorescent nucleotide addedwill have experienced three half-lives, thus reducing the fluorescentintensity of that base by about 95%. After four cycles, the intensitywill only be about 99% less than the original level. Therefore, thephotobleach pulse need not be applied long enough to completely bleachout the signal.

By continually reducing the fluorescence signal after the addition ofnucleotides, longer sequences are optionally sequenced than previouslymethods have allowed. For example, template nucleic acids of about 100or more, about 500 or more, about 1000 or more, about 2000 or more,about 10,000 or more, or about 50,000 or more nucleotides are optionallysequenced. Furthermore, since the fluorescence signal is photobleachedwith each nucleotide addition, with every other nucleotide addition,with every fifth nucleotide addition, or the like, the signal is readwith at least about 70% accuracy, at least about 80% accuracy, at leastabout 90% accuracy, or at least about 95% accuracy even when the nucleicacid template is about 500 or more, about 1000 or more, about 2000 ormore, about 10,000 or more, or about 50,000 or more nucleotides inlength.

In some embodiments, a build up of fluorescent signal as subsequentnucleotides are added is counteracted by using a combination offluorescently labeled nucleotides and non-labeled nucleotides. Forexample a low concentration of labeled nucleotides are added incombination with non-labeled nucleotides, e.g., in a 1/1000 ratio.Therefore, when a nucleotide is added, the small percentage of labelednucleotides added is detected. The non-labeled nucleotides do notinterfere with or contribute to signal buildup but are continuouslyelongated and available for subsequent additions. The read length for anucleic acid is thereby extended as in the photobleaching describedabove.

In other embodiments, non-labeled nucleotides are used in combinationwith labeled chain terminating nucleotides to increase read length. Theeventual read length typically depends on the efficiency with whichstrands are extended. For example, a 99% efficiency leads to a 64%reduction in signal after 100 cycles. If the incorporation of onefluorescent nucleotide reduces the efficiency of the next nucleotide(natural or fluorescent), then the read length is further compromised.One scheme to decrease the effect of incorporation efficiency after alabeled nucleotide is added is to use strand-terminating nucleotides forthe labeled nucleotides and mix them at low concentration withnon-labeled, non-terminating nucleotides, e.g., in a 1 to 1000 ratio.Therefore, only a small fraction of primer molecules are labeled andonly a small fraction are terminated. The remainder are continuouslyextended and detected. Therefore, the signal is not reduced and the readlength is extended.

V. Sequencing by Intercalation

Sequencing by synthesis using intercalating dyes for detection providesa way to measure an increased fluorescent signal whenever a nucleotideis incorporated into a nucleic acid chain, e.g., a primer strand. Thetemplate, primer and sequencing reagents, e.g., polymerase andnucleotides, are incubated in the presence of an intercalating dye. Whena nucleotide is incorporated into a primer strand, it extends thedouble-stranded region of the nucleic acid, and the intercalating dyeinserts or intercalates into that extended double stranded region.Therefore, whenever a nucleotide is added the signal is increased. Usingthis method of detection allows naturally-occurring nucleotides, e.g.,non-labeled nucleotides, to be used in the synthesis reactions.

A nucleic acid template and primer are hybridized according toprocedures well known in the art and as described above, resulting in adouble stranded region. For example, a nucleic acid template is attachedto a particle array, e.g., comprising ceramic beads. The primer ishybridized to the template strand, forming a double stranded region.

The hybridized template/primer is incubated with one of a series ofnucleotides, e.g., A, C, G, T, U, or the like, and an intercalator,e.g., an intercalating dye. The nucleotides are optionally unlabelednucleotides. For example, incubation optionally occurs by flowing thenucleotides across the particle array or flowing the particle arraythrough the nucleotides. The nucleotides and intercalator are optionallyadded together or separately. Addition of a nucleotide, e.g., if thenucleotide added is complementary to the template strand, results in anextended double-stranded region and the intercalating dye intercalatesor inserts itself into that region. The intercalating dye is thendetected to determine if the nucleotide was added. If an increase inintercalation is detected, e.g., by an increase in signal due to anadditional intercalating dye molecule in each template/primer strand, anucleotide was added. If no increase is detected, the nucleotide was notadded and the sequence is performed again with a different nucleotide,e.g., until the complementary base is determined. The templates andprimers are optionally rinsed after the addition of a nucleotide, thusremoving any unincorporated nucleotides from the reaction mixture eitherbefore or after detection.

In one embodiment, photobleaching is used, as described above, tophotobleach or reduce the fluorescence of the intercalators alreadypresent within the double-stranded region. Any added or additionalsignal detected is indicative of an additional stacked base in a doublestranded region. Alternatively, the intercalators remain intercalatedinto the stacked bases of the template and extended primer and newlyincorporated nucleotides are detected by measuring an increase in thefluorescent signal due to added intercalators.

Intercalating dyes typically intercalate into a double helix at the rateof 1 intercalator per about 4 to about 5 bases. However, in the presenceof many template/primer molecules and with the random intercalation ofthe dye, i.e., not specific to any to the sequence, the signal obtainedis typically only reduced by a factor of about 5 of the possible signalif every base is intercalated with the dye. This level of intercalationis easily detected, thus providing a new sequencing detection method.

The intercalators of the present invention are typically intercalatingdyes including, but not limited to, ethidium, ethidium bromide, anacridine dye, an intercalating nucleic acid stain, a cyanine dye, suchas SYBR green, proflavin, propidium iodide, acriflavin, proflavin,actinomycin, anthracyclines, or nogalamycin. The intercalators of thepresent invention typically comprise a detectable moiety, e.g., a labelas described above. For more information on possible intercalators, see,e.g., Handbook of Fluorescent Probes and Research Chemicals, RichardHaugland, Sixth Edition, Molecular Probes, Eugene Oregon (1996) andhttp:www.probes.com/handbook/sections/2300html (on-line 1999 version ofthe Handbook of Fluorescent Probes and Research Chemicals Sixth Editionby Molecular Probes, Inc.) (Molecular Probes, 1999).

The intercalating dye is optionally present in the reaction buffer oradded to the reaction as needed, e.g., after addition of a nucleotide tothe primer. For example in a microfluidic device, an intercalator isoptionally flowed across the template/primer molecules by application ofpressure or by electrokinetic gradients, e.g., by reverseelectrophoresis.

VI. Sequencing a Nucleic Acid in a Microfluidic Device

Any of the above sequencing technologies or any other known sequencingtechniques are optionally practiced in a microfluidic device, e.g., amicrofluidic device comprising bead arrays. The devices are optionallyfabricated to comprise nucleotide analogs as described above, as well asother sequencing reagents, such as intercalating dyes, fluorescentnucleotides, phosphate nucleotides, carbamate nucleotides, phosphatases,reducing agents, and the like. The reagents are optionally stored withinthe reservoirs of the devices, as described below, accessed through acapillary channel, e.g., from a microwell plate, or supplied on particlearrays.

Particle arrays are used, e.g., to immobilize a set of nucleic acidtemplates for sequencing. The template and/or primer are optionallyattached to a set of particles and positioned in or flowed through amicrofluidic device. For example, a porous barrier is used to immobilizethe particles (comprising nucleic acid templates and primers) within amicrofluidic channel. Reagents are then flowed across the particles tocontact the template and primer and sequence the nucleic acid template.In addition, used or spent reagents, e.g., unincorporated nucleotides orcleaved blocking groups, are washed from the channel while maintainingthe elongated nucleic acid in place, e.g., for another sequencing cycle.Particle arrays are discussed in more detail in U.S. Ser. No.60/128,643, filed Apr. 9, 1999 and in co-filed application,“Manipulation of Microparticles in Microfluidic Systems,” by Mehta etal., Attorney Docket No. 01-068-30. Microfluidic devices are describedbelow and in a number of patents and publications by the inventors andtheir co-workers. These publications are also described below.

The bead technology useful in the present invention typically usesarrays of particles, e.g., flowed through the channels of a microfluidicdevice. An “ordered array of a plurality of sets of particles” is anarray of particle sets (each particle set is constituted of similar oridentical particle “members” or “types”) having a spatial arrangement.The spatial arrangement of particle sets can be selected or random. In apreferred embodiment, the spatial arrangement is selected. Thearrangement can be known or unknown. In a preferred embodiment, thespatial arrangement of particle sets is known. A “set” of particles is agroup or “packet” of particles having similar or identical constituents.

The particles are typically flowed through the capillaries ormicrofluidic devices of the invention, e.g., to provide sequencingreagents or to contact nucleic acid templates and primers to performsequencing reactions. A “particle movement region” is a region of amicroscale element in which the particles are moved. A “fluid movementregion” is a region of a microscale element in which fluidic componentsare moved. As discussed supra, fluidic and particulate elements aremoved by any of a variety of forces, including capillary, pressure,electrokinetic and the like.

A “particle retention region” is a region of a microscale element inwhich particles can be localized, e.g., by placing a physical barrier orporous matrix within or proximal to the retention region, by applicationof magnetic or electric fields, by application of pressure, or the like.For example, a porous matrix optionally comprises a fixed set ofparticles, e.g., 186 μm particles, within a microchannel.

A train of reagents (i.e., an ordered or semi-ordered arrangement offluidic reagents in a channel) comprising a plurality of sequencingreagents is flowed across the first set of particles, or the first setof particles is flowed through the reagent train, depending on theapplication. This results in contacting the at least one set of nucleicacid templates with the plurality of sequencing reagents. Signalsresulting from exposure of the first set of particles to the reagenttrain are selected, thereby providing a portion of sequence of thenucleic acid template. For example, the reagent train can include apolymerase, a sufurylase, an apyrase, an inorganic phosphate, ATP, athermostable polymerase, luciferin, luciferase, an endonuclease, anexonuclease, Mg⁺⁺, a molecular crowding agent, a buffer, a dNTP, a dNTPanalog, a fluorescent nucleotide, a chain terminating nucleotide, areversible chain terminating nucleotide, a phosphatase, a reducingagent, an intercalator, a salt, DTT, BSA, a detergent (e.g., triton® ortween®), chemicals to inhibit or enhance EO flow (e.g., polyacrylamide),or any other sequencing reagent of interest.

The number of ordered sets constituting the array depends on theselected application. For example, as discussed in more detail herein,one exemplar array for sequencing nucleic acids comprises about 2, 3, or4 sets of particles (e.g., beads, cells, microspheres, etc.). In otherimplementations, 5, 10, 50, 100, 500, 1000, 5,000, 10,000, 50,000 oreven 100,00 or more different sets of particles can be present in thearrays. The precise number of particles in an array depends on theintended use of the array.

The array components (i.e., particles) of the arrays of the inventioncan be essentially any discreet material, which can be flowed through amicroscale system. Example particles include beads and biological cells.For example, polymer beads (e.g., polystyrene, polypropylene, latex,nylon and many others), silica or silicon beads, clay or clay beads,ceramic beads, glass beads, magnetic beads, metallic beads, inorganiccompound beads, and organic compound beads can be used. An enormousvariety of particles are commercially available, e.g., those typicallyused for chromatography (see, e.g., the 1999 Sigma “Biochemicals andReagents for Life Sciences Research” Catalog from Sigma (Saint Louis,Mo.), e.g., pp. 1921-2007; The 1999 Suppleco “Chromatography Products”Catalogue, and others), as well as those commonly used for affinitypurification (e.g., Dynabeads™ from Dynal, as well as many derivitizedbeads, e.g., various derivitized Dynabeads™ (e.g., the various magneticDynabeads™, which commonly include coupled reagents) supplied e.g., byPromega, the Baxter Immunotherapy Group, and many other sources).

The array particles can have essentially any shape, e.g., spherical,helical, spheroid, rod-shaped, cone-shaped, cubic, polyhedral, or acombination thereof (of course they can also be irregular, as is thecase for cell-based particles). In addition, the particles can be avariety of sizes. Typically, the particles are about 0 1 μm to about 500μm. Alternatively, the particles are about 0.5 μm to about 50 μm orabout 1 μm to about 20 μm. Particles are optionally coupled to reagents,affinity matrix materials, or the like, e.g., nucleic acid synthesisreagents, peptide synthesis reagents, polymer synthesis reagents,nucleic acids, nucleotides, nucleobases, nucleosides, peptides, aminoacids, monomers, cells, biological samples, synthetic molecules, orcombinations thereof. Particles optionally serve many purposes withinthe arrays, including acting as blank particles, dummy particles,calibration or marker particles, capture devices for low concentrationreagents, sample particles, reagent particles and test particles.

The particles within the arrays of the invention can present a solid orsemi-solid surface for any of a variety of linking chemistries, allowingthe incorporation of biological and chemical components of interest intothe particle members of the arrays. A wide variety of organic andinorganic polymers, both natural and synthetic may be employed as thematerial for the solid surface. Illustrative polymers includepolyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones,polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and thelike. Other materials that may be employed include papers, ceramics,such as glass, metals, metalloids, semiconductive materials, cements, orthe like. In addition, substances that form gels, such as proteins(e.g., gelatins), lipopolysaccharides, silicates, agarose and are alsooptionally used.

A wide variety of linking chemistries are available for linkingmolecules to a wide variety of solid or semi-solid particle supportelements. These chemistries can be performed in situ (i.e., in themicrofluidic system, by flowing appropriate reagents, e.g., nucleicacids, proteins, and samples present in low concentrations, into contactwith the particles, or vice-versa), or outside of a microfluidicenvironment, e.g., prior to introduction of the particles into themicrofluidic system. It is impractical and unnecessary to describe allof the possible known linking chemistries for linking molecules to asolid support. It is expected that one of skill can easily selectappropriate chemistries, depending on the intended application.

In one preferred embodiment, the particles of the invention comprisesilicate elements (e.g., glass or silicate beads). An array ofsilicon-based molecules appropriate for functionalizing surfaces iscommercially available. See, for example, Silicon Compounds Registry andReview, United Chemical Technologies, Bristol, Pa. Additionally, the artin this area is very well developed and those of skill will be able tochoose an appropriate molecule for a given purpose. Appropriatemolecules can be purchased commercially, synthesized de novo, or formedby modifying an available molecule to produce one having the desiredstructure and/or characteristics.

The substrate linker attaches to the solid substrate through any of avariety of chemical bonds. For example, the linker is optionallyattached to the solid substrate using carbon-carbon bonds, for examplevia substrates having (poly)trifluorochloroethylene surfaces, orsiloxane bonds (using, for example, glass or silicon oxide as the solidsubstrate). Siloxane bonds with the surface of the substrate are formedin one embodiment via reactions of derivatization reagents bearingtrichlorosilyl or trialkoxysilyl groups. The particular linking group isselected based upon, e.g., its hydrophilic/hydrophobic properties wherepresentation of an attached polymer in solution is desirable. Groupsthat are suitable for attachment to a linking group include amine,hydroxyl, thiol, carboxylic acid, ester, amide, isocyanate andisothiocyanate. Preferred derivatizing groups includeaminoalkyltrialkoxysilanes, hydroxyalkyltrialkoxysilanes,polyethyleneglycols, polyethyleneimine, polyacrylamide, polyvinylalcoholand combinations thereof.

VII. Integrated Systems and Kits

The microfluidic devices of the present invention are used to performsequencing by incorporation or sequencing by synthesis. The devicesgenerally comprise a body structure with microscale channels or othercavities fabricated therein. Typically, the device includes reagentsources, a main channel, and a detection region. For example, tosequence a nucleic acid, templates and primers are typically flowedthrough a main channel of a microfluidic device and immobilized withinthe channel. Alternatively, microfluidic devices are provided withtemplates and primers immobilized therein. Sequencing reagents, e.g.,polymerases and nucleotides, are flowed from reagent sources, e.g.,reservoirs or microwell plates, to contact the template and primer.Complementary nucleotides are incorporated and detected, e.g., in adetection region proximal to the immobilized templates and primers. Thesteps are iteratively repeated to provide, e.g., an entire templatesequence or portion thereof.

For example, a device typically comprises a main channel, which mainchannel comprises the template and primer, e.g., on a particle array.The main channel typically comprises a particle retention region and aflow region. For example a particle array is fixed in the main channelin the particle retention region and reagents are flowed through theflow region and across the particle array to sequence a nucleic acidtemplate. For example, FIG. 1 provides a particle array stacked or fixedin a microscale channel, e.g., by a porous matrix. Reagents are flowedthrough the channel and across the particle array. Unincorporatednucleotides are optionally washed out of the channel, e.g., through theporous matrix. The main channel or microscale cavity is used, e.g., tomix or incubate two or more reagents, to react two or more reagents, todilute reagents, to separate various components, and the like.Typically, the reservoirs or sources of reagents are fluidly coupled tothe main channel so that reagents are optionally introduced into themain channel from the reservoirs.

Reagent sources are typically fluidly coupled to the main channel. Thereagent sources are typically reservoirs or wells fluidly coupled to themain channel for adding, removing, or storing the various reagents ofinterest, e.g., sequencing reagents. Alternatively, the reagent sourcecomprises a sipper capillary fluidly coupled to the main channel and toa reagent source, e.g., a microwell plate. A train of reagents isoptionally stored in a microwell plate, which is then accessed by thesipper capillary for addition into the device.

A detection region is typically included in the devices of the presentinvention for the detection of labeled compounds. For example, thenucleic acids are optionally flowed through a detection region, e.g., aregion of the main channel, after addition of a nucleic acid.Alternatively, the nucleic acids, e.g., attached to particle arrays, arefixed in the channel within the detection region.

The detection region is optionally a subunit of a channel, or itoptionally comprises a distinct channel that is fluidly coupled to theplurality of channels in the microfluidic device, e.g., to the mainchannel. The detection region typically includes a window at which asignal is monitored. The window typically includes a transparent coverallowing visual or optical observation and detection of the assayresults, e.g., observation of a colorimetric or fluorometric signal orlabel. Examples of suitable detectors are well known to those of skillin the art and are discussed in more detail below.

The above channel regions are fluidly coupled to each other and tovarious pressure sources and/or electrokinetic sources. Fluidicmaterials, such as polymerase solutions, nucleotides, reducing agents,sequencing reagents, and the like, are typically transported through theinterconnected channel system by the application of pressure and/orelectrokinetic forces to the fluid materials in the channels. Therefore,various pressure sources and electrokinetic controllers are optionallycoupled to the devices of the invention.

Typically, the pressure sources are applied at channel ends. For examplea waste well is optionally placed at one end of a main channel with asample source at the other end. A pressure source applied at the wastewell is optionally used to draw fluid into the channel. For example, avacuum source may be fluidly coupled to the device at a waste reservoirlocated at the end of the main channel. The vacuum optionally draws anyexcess, or unused material, e.g., unincorporated nucleotides, into thewaste reservoir to which the vacuum source is fluidly coupled. Forexample the vacuum pulls fluid through a porous matrix into a wastereservoir. Alternatively, a positive pressure source is fluidly coupledto a sample well or reservoir at one end of a main channel. The pressurethen forces the material into and through the main channel. The vacuumsource draws fluid into the main channel for mixing or reacting withother reagents.

Alternatively, electrokinetic forces, e.g., high or low voltages, areapplied at reservoirs to introduce materials into the channels ortransport materials through the channels. For example, voltage gradientsapplied across a separation channel are used to move fluid down thechannel, thus separating the components of the material as they movethrough the channel at different rates. In other embodiments,centrifugal force is used to flow reagents through channels.

One embodiment of the present devices is illustrated in FIG. 1. Asshown, the system comprises main channel 105, which is optionally acapillary or a channel in a microfluidic device. To sequence a nucleicacid, various reagents are added into main channel 105. For example, atemplate and primer are introduced into main channel 105 from amicrowell plate. The template and primer are then captured and retainedby particle set 115, which is held in place by particle retentionelement 110. Other reagents used in the sequencing are introduced intomain channel 105 also. For example, a polymerase solution and a mixtureof nucleotides are introduced into main channel 102 from, e.g., amicrowell plate or a reservoir located within the microfluidic device.In the presence of the polymerase, one or more nucleotides are added tothe nucleic acid primer to form an extended nucleic acid. Unincorporatednucleotides are washed from the channel, e.g., flowed through porousparticle retention element 110. The newly incorporated nucleotides aredetected, e.g., by fluorescence detection, by a detector positionedproximal to particle set 115.

In another embodiment, nucleic acids are sequenced in a multi-channeldevice 800 as shown in FIG. 8. For example, template and primer areloaded into main channel 850 through sipper capillary 801, e.g., usingpressure control at the sipper and/or wells 810-845. Nucleotides andbuffer are stored, e.g., in reagent wells 810-845, and sequentiallyintroduced into main channel 850. For example, dATP is flowed from well845 into channel region 890 and then into main channel 850, e.g., addingdATP to the primer strand if it is complementary to the template. Thechannel is then rinsed with buffer, e.g., from well 840, to removeunincorporated dATP, e.g., into waste well 805. The remainingnucleotides, e.g., dCTP, dTTP, and dGTP, are added in the samesequential manner, e.g., until the template or a desired portion thereofis sequenced. For example, wells 810-835 are optionally used to add theremaining nucleotides, through channel regions 860-880, into mainchannel 850 to contact the template and primer.

In some embodiments, one or more of the components are attached to oneor more sets of particles, which are flowed through the device in thesame manner. For example, in a preferred embodiment, the template andprimer are typically attached to a set of particles which is flowed intomain channel 850 and immobilized therein. The nucleotides and buffersare flowed across the set of particles and unincorporated nucleotidesare removed, e.g., into waste well 805. A detector is optionally placedproximal to main channel 850 to detect incorporated nucleotides. Any ofthe above described sequencing by synthesis methods are optionallyperformed in this manner.

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few or oneparticular operation, e.g., a sequencing using particle arrays, it willbe readily appreciated from this disclosure that the flexibility ofthese systems permits easy integration of additional operations intothese devices. For example, the devices and systems described willoptionally include structures, reagents and systems for performingvirtually any number of operations both upstream and downstream from theoperations specifically described herein. Such upstream operationsinclude sample handling and preparation operations, e.g., cellseparation, extraction, purification, amplification, cellularactivation, labeling reactions, dilution, aliquotting, and the like.Similarly, downstream operations may include similar operations,including, e.g., separation of sample components, labeling ofcomponents, assays and detection operations, electrokinetic orpressure-based injection of components, or the like. The devices andsystems used for the above assays are described below.

Microfluidic Devices Generally

A variety of microscale systems are optionally adapted to the presentinvention by incorporating particle arrays, polymerases, templates,primers, sequencing reagents, and the like. A variety of microfluidicdevices are optionally adapted for use in the present invention, e.g.,by designing and configuring the channels as discussed below. Theinventors and their co-workers have provided a variety of microfluidicsystems in which the arrays of the invention can be constructed andsequencing reactions carried out. For example, Ramsey WO 96/04547provides a variety of microfluidic systems. See also, Ramsey et al.(1995), Nature Med. 1(10): 1093-1096; Kopf-Sill et al. (1997)“Complexity and performance of on-chip biochemical assays,” SPIE2978:172-179 February 10-11; Bousse et al. (1998) “Parallelism inintegrated fluidic circuits,” SPIE 3259:179-186; Chow et al. U.S. Pat.No. 5,800,690; Kopf-Sill et al. U.S. Pat. No. 5,842,787; Parce et al.,U.S. Pat. No. 5,779,868; Parce, U.S. Pat. No. 5,699,157; U.S. Pat. No.5,852,495 (J. Wallace Parce) issued Dec. 22, 1998; U.S. Pat. No.5,869,004 (J. Wallace Parce et al.) issued Feb. 9, 1999, U.S. Pat. No.5,876,675 (Colin B. Kennedy) issued Mar. 2, 1999; U.S. Pat. No.5,880,071 (J. Wallace Parce et al.) issued Mar. 9, 1999; U.S. Pat. No.5,882,465 (Richard J. McReynolds) issued Mar. 16, 1999; U.S. Pat. No.5,885,470 ( J. Wallace Parce et al.) issued Mar. 23, 1999; U.S. Pat. No.5,942,443 (J. Wallace Parce et al.) issued Aug. 24, 1999; U.S. Pat. No.5,948,227 (Robert S. Dubrow) issued Sep. 7, 1999; U.S. Pat. No.5,955,028 (Calvin Y. H. Chow) issued Sep. 21, 1999; U.S. Pat. No.5,957,579 (Anne R. Kopf-Sill et al.) issued Sep. 28, 1999; U.S. Pat. No.5,958,203 (J. Wallace Parce et al.) issued Sep. 28, 1999; U.S. Pat. No.5,958,694 (Theo T. Nikiforov) issued Sep. 28, 1999; and U.S. Pat. No.5,959,291 (Morten J. Jensen) issued 09/28/199; Parce et al. WO 98/00231;Parce et al. WO 98/00705; Chow et al. WO 98/00707; Parce et al. WO98/02728; Chow WO 98/05424; Parce WO 98/22811; Knapp et al., WO98/45481; Nikiforov et al. WO 98/45929; Parce et al. WO 98/46438; Dubrowet al., WO 98/49548; Manz, WO 98/55852; WO 98/56505; WO 98/56956; WO99/00649; WO 99/10735; WO 99/12016; WO 99/16162; WO 99/19056; WO99/19516; WO 99/29497; WO 99/31495; WO 99/34205; WO 99/43432; and WO99/44217; U.S. Pat. No. 5,296,114; and e.g., EP 0 620 432 A1; Seiler etal. (1994) Mitt Gebiete Lebensm. Hyg. 85:59-68; Seiler et al. (1994)Anal. Chem. 66:3485-3491; Jacobson et al. (1994) “Effects of InjectionSchemes and Column Geometry on the Performance of MicrochipElectrophoresis Devices” Anal. Chem. 66: 66. 1107-1113; Jacobsen et al.(1994) “Open Channel Electrochromatograpy on a Microchip” Anal. Chem.66:2369-2373; Jacobsen et al. (1994) “Precolumn Reactions withElectrophoretic Analysis Integrated on Microchip” Anal. Chem.66:4127-4132; Jacobsen et al. (1994) “Effects of Injection Schemes andColumn Geometry on the Performance of Microchip ElectrophoresisDevices.” Anal. Chem. 66:1107-1113; Jacobsen et al. (1994) “High SpeedSeparations on a Microchip.” Anal. Chem. 66:1114-1118; Jacobsen andRamsey (1995) “Microchip electrophoresis with sample stacking”Electrophoresis 16:481-486; Jacobsen et al. (1995) “Fused QuartzSubstrates for Microchip Electrophoresis” Anal. Chem. 67: 2059-2063;Harrison et al. (1992) “Capillary Electrophoresis and Sample InjectionSystems Integrated on a Planar Glass Chip.” Anal. Chem. 64:1926-1932;Harrison et al. (1993) “Micromachining a Miniaturized CapillaryElectrophoresis-Based Chemical Analysis System on a Chip.” Science 261:895-897; Harrison and Glavania (1993) “Towards MiniaturizedElectrophoresis and Chemical System Analysis Systems on Silicon: AnAlternative to Chemical Sensors.” Sensors and Actuators 10:107-116; Fanand Harrison (1994) “Micromachining of Capillary ElectrophoresisInjectors and Separators on Glass Chips and Evaluation of Flow atCapillary Intersections. Anal. Chem. 66: 177-184; Effenhauser et al.(1993) “Glass Chips for High-Speed Capillary Electrophoresis Separationswith Submicrometer Plate Heights” Anal. Chem. 65:2637-2642; Effenhauseret al. (1994) “High-Speed Separation of Antisense Oligonucleotides on aMicromachined Capillary Electrophoresis Device.” Anal. Chem.66:2949-2953; and Kovacs EP 0376611 A2.

The above devices, systems, features, and components are used in theintegrated systems described below, e.g., to sequence nucleic acids.

For example, the channel 105 in FIG. 1 is optionally used to sequence anucleic acid by synthesis. A set of templates and/or primers is attachedto particle set 115 and flowed through or positioned within channel 105.Particle retention element 110 is optionally used to immobilize particleset 115 comprising the templates and primers. Alternatively, particleset 115 is held in place by magnetic force or chemically attached to thesurface of channel 105. Sequencing reagents are typically flowed acrossparticle set 115. For example, dATP, dGTP, dTTP, and dCTP, e.g., eachcomprising a reversible chain terminating blocking group and labeledwith a distinguishable fluorescent label are flowed through channel 105to contact the template and primer on particle set 115. A nucleotide isincorporated into the primer, e.g., a nucleotide that is complementaryto the template nucleic acid, and detected. For example, unincorporatednucleotides are removed from the channel, e.g., by flowing bufferthrough the channel, and the identity of the incorporated nucleotide isdetermined based on the fluorescent signal measured, e.g., by a detectorpositioned proximal to particle set 115. Reagents are then flowedthrough channel 105 to remove the 3′-blocking groups so that additionalnucleotides are incorporated into the primer nucleic acid. For example,a reducing agent and/or a phosphatase is flowed through channel 105 toremove a phosphate blocking group. Alternatively, the blocking groupsare removed prior to detection and detection occurs downstream ofparticle retention element 110, e.g., as the removed and labeledblocking groups are flowed through the porous barrier formed by particleretention element 110.

In another embodiment, multiple particle sets are used in a microfluidicchannel to sequence one or more nucleic acid template by synthesis. FIG.6 illustrates capillary 605 comprising multiple particle sets 615, 617,and 619. In addition, particle retention element 610 optionallycomprises an immobilized particle set. In one aspect, each of particlesets 615-619 comprises a different nucleic acid template. Reagents areoptionally flowed through the channel as described above, therebysequencing each nucleic acid template, or the nucleotides are flowedthrough the channel in series. For example, fluorescent dCTP isoptionally flowed through channel 605 and incubated with thetemplate/primer nucleic acids. A buffer is typically flowed through thechannel to remove any unincorporated dCTP. Any incorporated dCTP remainsin the channel as part of the primer strand that is attached toimmobilized particle set 615, 617, or 619. Any incorporated dCTP is thendetected. For example, a detector is placed proximal to each of theparticle sets or a single detector scans across each particle set todetect any incoporated dCTP. The procedure is then repeated as eachnucleotide of interest is flowed across particle sets 615-619 todetermine the next nucleotide in the template sequence.

In some embodiments reagents are also optionally associated with orattached to a particle set. The reagents are optionally brought intocontact with templates and/or primers, e.g., templates and primersimmobilized on capillary or channel or walls, and removed from theparticles, e.g., by washing or by chemical cleavage. Once removed fromthe particle set, reagents, e.g., nucleotides are optionallyincorporated into the templates.

In another embodiment, hybridized template/primer sequences areimmobilized onto particle sets that are then flowed through variousreagents as illustrated by FIG. 5. Microfluidic device 500 comprisesmain channel 510 and reagent introduction channels 515-530 (as depicted,these are coupled to reagents for separate sequencing reactions, e.g.,comprising A, G, C, or T nucleotides). Sample train 531 comprising aplurality of samples, e.g., particle sets 535-550, is passed back andforth through intersections 560-590. Reagent from channels 515-530 isflowed across each sample (or selected samples) in train 531 as thetrain passes the corresponding coupled intersection.

A number of capillaries or channels as described above are optionallybanked together, e.g., as parallel channels in a microfluidic device, toprovide a high throughout system for sequencing nucleic acids. Aschematic of such a system is provided in FIG. 7. FIG. 7 shows aplurality of microtiter plates, e.g., plates 705, 710, and 715. Eachwell contains a set of particles comprising a nucleic acid template.Therefore, the system shown optionally comprises a plurality ofdifferent nucleic acid templates, e.g., about 500 or more, about 1000 ormore, or the like, that are optionally sequenced in a high throughputmanner. Additional microwell plates and channels are optionally used toprovide a greater number of templates. A plate of blank particle sets isalso optionally included, e.g., plate 720. The particle sets are loadedinto a set of capillaries or channels as shown by channel set 725. Thecapillaries optionally comprise capillaries or channels as shown in FIG.1, FIG. 5, FIG. 6, or the like. Multiple particle sets are flowedthrough each channel and sequenced. For example, 96 particle sets areoptionally loaded into each of 12 channels using 12 sipper capillaries,e.g., capillary set 740, or one sipper capillary fluidly coupled to eachof the 12 channels. The particle sets are typically retained in thecapillary or microchannel by a porous particle retention element, e.g.,a sintered glass frit, a set of epoxy coated particles, a constrictedchannel region, or the like. The particle retention element fixes orretains the particle sets, e.g., particle sets comprising nucleic acidtemplates, in the channel. The particle sets, e.g., templates, are thenoptionally exposed to a series or train of reagents. The reagents aretypically added through each channel, e.g., from another set ofmicrowell plates, to perform various assays, e.g., sequencing. A singlecontroller, e.g., controller 730 is optionally used to control fluidflow through sipper set 740 and channel set 725. One or more detector isused to monitor the particle packets in the channels as variousnucleotides are added. Alternatively, detectors are positioneddownstream of the channels to monitor the waste products, e.g., todetect a fluorescent label that has since been washed from the channels.For example, detection optionally occurs in detection region 735. Usinga system such as that shown in FIG. 7, one particle set is optionallyloaded in about one minute. Therefore 96 templates are optionallyanalyzed, e.g., sequenced, in 1.6 hours. Alternatively, particles withdifferent chemistries are arrayed sequentially in a single capillary anda template is flowed across the array, e.g., for sequencing.

Instrumentation

In the present invention, materials such as enzymes, nucleic acids,nucleotides, and nucleotide analogs, are optionally monitored and/ordetected so that presence of a product of interest can be detected or anactivity or concentration can be determined. For example, in asequencing reaction, one or more nucleotides are added to a growingnucleic acid chain. The nucleotides are typically detected as they areadded to the chain when sequencing by synthesis. Therefore, thenucleotides or nucleotide analogs are typically labeled as describedabove and detected using the instrumentation and integrated systemsdescribed below. In some embodiments, the nucleotides are not labeledand labeled intercalating dyes are used to detect addition ofnucleotides. Depending on the label signal measurements, decisions areoptionally made regarding subsequent fluidic operations, e.g., whetherto add a different nucleotide. For example, in some sequencingembodiments, a series of labeled nucleotides, e.g., ATP CTP, GTP, andTTP, is incubated with the template. The template is contacted with afirst nucleotide and unincorporated nucleotides are washed from thereaction mixture. If the nucleotide was added to the chain, it isdetected. If the nucleotide was not added, no signal is detected. If thesignal was detected, then the series of nucleotides begins again. If thenucleotide was not added, then the next nucleotide in the series isadded until the identity of that position in the template is determined.

The systems described herein generally include microfluidic devices, asdescribed above, in conjunction with additional instrumentation forcontrolling fluid transport, flow rate and direction within the devices,detection instrumentation for detecting or sensing results of theoperations performed by the system, processors, e.g., computers, forinstructing the controlling instrumentation in accordance withpreprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format.

Fluid Direction System

A variety of controlling instrumentation is optionally utilized inconjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluidic materials and/ormaterials within the devices of the present invention, e.g., bypressure-based, electrokinetic, magnetic, or centrifugal control orcombinations thereof. For example electrophoretic control systems areused to transport particle arrays and reagents through various channelregions. Alternatively, magnetic filed are used to transport magneticbeads or particle arrays, e.g., magnetic beads comprising sequencingreagents.

In the present system, the fluid direction system controls thetransport, flow and/or movement of a template, a primer, a particlearray, a series of nucleotides, or the like, through the microfluidicdevice. For example, the fluid direction system optionally directs themovement of a template and a primer through a main channel, in which thetemplate and primer are incubated and hybridized. Sequencing reagentsare also optionally added, e.g., buffers, salts, nucleotides, enzymes,and the like. The reagents mix and/or react with the template and theprimer in the main channel.

In general, nucleic acids, particle arrays, nucleotides, and othercomponents can be flowed in a microscale system by electrokinetic(including either electroosmotic or electrophoretic) techniques, and/orusing pressure-based flow mechanisms, or combinations thereof.

Electrokinetic material transport systems or electrokinetic controllersare used in the present invention to provide movement of particle arraysand sequencing reagents through microfluidic channels. “Electrokineticmaterial transport systems,” as used herein, include systems thattransport and direct materials within a microchannel and/or microchambercontaining structure, through the application of electrical fields tothe materials, thereby causing material movement through and among thechannel and/or chambers, i.e., cations will move toward a negativeelectrode, while anions will move toward a positive electrode. Forexample, movement of fluids toward or away from a cathode or anode cancause movement of proteins, enzymes, peptides, modulators, etc.suspended within the fluid. Similarly, the components, e.g., proteins,peptides, amino acids, enzymes, etc. can be charged, in which case theywill move toward an oppositely charged electrode (indeed, in this case,it is possible to achieve fluid flow in one direction while achievingparticle flow in the opposite direction). In this embodiment, the fluidcan be immobile or flowing and can comprise a matrix as inelectrophoresis.

Typically, the electrokinetic material transport and direction systemsof the invention rely upon the electrophoretic mobility of chargedspecies within the electric field applied to the structure. Such systemsare more particularly referred to as electrophoretic material transportsystems. For example, in the present system separation of a mixture ofcomponents into its individual components typically occurs byelectrophoretic separation. For electrophoretic applications, the wallsof interior channels of the electrokinetic transport system areoptionally charged or uncharged. Typical electrokinetic transportsystems are made of glass, charged polymers, and uncharged polymers. Theinterior channels are optionally coated with a material that alters thesurface charge of the channel.

A variety of electrokinetic controllers and systems which are optionallyused in the present invention are described, e.g., in U.S. Pat. No.5,858,195, by Ramsey issued Jan. 12, 1999, Parce et al. WO 98/46438 andDubrow et al., WO 98/49548, as well as a variety of other referencesnoted herein.

Use of electrokinetic transport to control material movement ininterconnected channel structures was described, e.g., in WO 96/04547and U.S. Pat. No. 5,858,195 to Ramsey. An exemplary controller isdescribed in U.S. Pat. No. 5,800,690. Modulating voltages areconcomitantly applied to the various reservoirs to affect a desiredfluid flow characteristic, e.g., continuous or discontinuous (e.g., aregularly pulsed field causing the sample to oscillate direction oftravel) flow of labeled products in one or more channels toward adetection region or waste reservoir.

Other methods of transport are also available for situations in whichelectrokinetic methods are not desirable. For example, sampleintroduction and reaction are best carried out in a pressure-basedsystem to avoid electrokinetic biasing during sample mixing and highthroughput systems typically use pressure induced sample introduction.Pressure based flow is also desirable in systems in which electrokinetictransport is also used. For example, pressure based flow is optionallyused for introducing and reacting reagents in a system in which theproducts are electrophoretically separated.

Pressure can be applied to microscale elements, e.g., to a channel,region, or reservoir, to achieve fluid movement using any of a varietyof techniques. Fluid flow (and flow of materials suspended orsolubilized within the fluid, including cells or other particles) isoptionally regulated by pressure based mechanisms such as those basedupon fluid displacement, e.g., using a piston, pressure diaphragm,vacuum pump, probe, or the like, to displace liquid and thereby raise orlower the pressure at a site in the microfluidic system. The pressure isoptionally pneumatic, e.g., a pressurized gas, or uses hydraulic forces,e.g., pressurized liquid, or alternatively, uses a positive displacementmechanism, i.e., a plunger fitted into a material reservoir, for forcingmaterial through a channel or other conduit, or is a combination of suchforces.

In some embodiments, a vacuum source is applied to a reservoir or wellat one end of a channel to draw a fluidic material through the channel.Pressure or vacuum sources are optionally supplied external to thedevice or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel. Examples of microfabricated pumps havebeen widely described in the art. See, e.g., published InternationalApplication No. WO 97/02357. A vacuum applied to a main channel isoptionally used to drive fluid flow. For example, a vacuum is used todraw fluid, e.g., unincorporated nucleotides, through a porous barrierand into a waste reservoir.

Hydrostatic, wicking and capillary forces are also optionally used toprovide fluid pressure for continuous fluid flow of materials such asenzymes, substrates, modulators, or protein mixtures. See, e.g., “METHODAND APPARATUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USINGPRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION,” by Alajoki etal., U.S. Pat. No. 6,416,642. In these methods, an adsorbent material orbranched capillary structure is placed in fluidic contact with a regionwhere pressure is applied, thereby causing fluid to move towards theadsorbent material or branched capillary structure. The capillary forcesare optionally used in conjunction with the electrokinetic orpressure-based flow in the present invention. The capillary action pullsmaterial through a channel. For example a wick is optionally added to,e.g., main channel 105, to aid fluid flow by drawing the reactantsand/or products, e.g., unincorporated nucleotides, through the channel,e.g., toward a waste reservoir.

Mechanisms for reducing adsorption of materials during fluid-based floware described in “PREVENTION OF SURFACE ADSORPTION IN MICROCHANNELS BYAPPLICATION OF ELECTRIC CURRENT DURING PRESSURE-INDUCED FLOW” filed05/11/1999 by Parce et al., Attorney Docket Number 01-78-0. In brief,adsorption of cells, components, proteins, enzymes, and other materialsto channel walls or other microscale components during pressure-basedflow can be reduced by applying an electric field such as an alternatingcurrent to the material during flow.

Mechanisms for focusing labeling reagents, enzymes, modulators, andother components into the center of microscale flow paths, which isuseful in increasing assay throughput by regularizing flow velocity,e.g., in pressure based flow, is described in “FOCUSING OFMICROPARTICLES IN MICROFLUIDIC SYSTEMS” by H. Garrett Wada et al. U.S.Ser. No. 60/134,472, filed May 17, 1999. In brief, sample materials arefocused into the center of a channel by forcing fluid flow from opposingside channels into the main channel comprising the cells, or by otherfluid manipulations.

In an alternate embodiment, microfluidic systems can be incorporatedinto centrifuge rotor devices, which are spun in a centrifuge. Fluidsand particles travel through the device due to gravitational andcentripetal/centrifugal pressure forces. For example, unincorporatednucleotides are optionally removed from a set of particles comprising anucleic acid template and primer using centrifugal force.

In addition to transport through the microfluidic system, the inventionalso provides for introduction of sample or reagents, e.g., enzymes,nucleotides, nucleic acids, particle sets, and the like, into themicrofluidic system. Sources of samples, mixtures of components, andreagents, e.g., enzymes, substrates, labeling reagents, and the like,are fluidly coupled to the microchannels noted herein in any of avariety of ways. In particular, those systems comprising sources ofmaterials set forth in Knapp et al. “Closed Loop Biochemical Analyzers”(WO 98/45481; PCT/US98/06723) and Parce et al. “High ThroughputScreening Assay Systems in Microscale Fluidic Devices” WO 98/00231 and,e.g., in 60/128,643 filed Apr. 4, 1999, entitled “Manipulation ofMicroparticles In Microfluidic Systems,” by Mehta et al. are applicable.

In these systems, a “pipettor channel” (a channel in which componentscan be moved from a source to a microscale element such as a secondchannel or reservoir) is temporarily or permanently coupled to a sourceof material. The source can be internal or external to a microfluidicdevice comprising the pipettor channel. Example sources includemicrowell plates, membranes or other solid substrates comprisinglyophilized components, wells or reservoirs in the body of themicroscale device itself and others.

Alternative sources include a well disposed on the surface of the bodystructure comprising the template, primer, sequencing reagent, or thelike, a reservoir disposed within the body structure comprising thenucleic acid sample or template, sequencing reagents; a containerexternal to the body structure comprising at least one compartmentcomprising a template, primer, sequencing reagent, particle set, or thelike, or a solid phase structure comprising the template, primer,sequencing reagents, particle sets, or the like in lyophilized orotherwise dried form.

A loading channel region is optionally fluidly coupled to a pipettorchannel with a port external to the body structure. The loading channelcan be coupled to an electropipettor channel with a port external to thebody structure, a pressure-based pipettor channel with a port externalto the body structure, a pipettor channel with a port internal to thebody structure, an internal channel within the body structure fluidlycoupled to a well on the surface of the body structure, an internalchannel within the body structure fluidly coupled to a well within thebody structure, or the like.

The integrated microfluidic system of the invention optionally includesa very wide variety of storage elements for storing samples and reagentsto be assessed. These include well plates, matrices, membranes and thelike. The reagents are stored in liquids (e.g., in a well on amicrotiter plate), or in lyophilized form (e.g., dried on a membrane orin a porous matrix), and can be transported to an array component,region, or channel of the microfluidic device using conventionalrobotics, or using an electropipettor or pressure pipettor channelfluidly coupled to a region or channel of the microfluidic system. Suchreagents include, but are not limited to, labeling reagents, e.g.,enzymes, e.g., phosphatases and polymerases, sequencing reagents,nucleic acids, primers, and the like.

Typically, the fluid controller systems are appropriately configured toreceive or interface with a microfluidic device or system element asdescribed herein. For example, the controller and/or detector,optionally includes a stage upon which the device of the invention ismounted to facilitate appropriate interfacing between the controllerand/or detector and the device. Typically, the stage includes anappropriate mounting/alignment structural element, such as a nestingwell, alignment pins and/or holes, asymmetric edge structures (tofacilitate proper device alignment), and the like. Many suchconfigurations are described in the references cited herein.

Detection System

The devices herein optionally include signal detectors, e.g., whichdetect fluorescence, phosphorescence, radioactivity, pH, charge,absorbance, luminescence, temperature, magnetism, color, or the like.Fluorescent detection is especially preferred. For example, nucleotidesthat have been added to a growing chain of nucleotides are optionallydetected by fluorescent photobleaching

The detector(s) optionally monitors one or a plurality of signals fromthe nucleic acid template, which is typically immobilized in amicrofluidic channel or capillary. For example, the detector optionallymonitors an optical signal that corresponds to a labeled component, suchas a labeled nucleotide, e.g., that has been added to a templateimmobilized in a channel. For example, in FIG. 1, templates and primersare typically attached to particle set 115, which is immobilized inchannel 105 using particle retention element 110. A detector placedproximal to particle set 115, detects each nucleotide as it isincorporated into the nucleic acid chain.

In another embodiment, a nucleotide is added to a growing chain and alabeled 3′-blocking group is removed from the chain to allow for furtherelongation. Detection optionally occurs before or after removal of the3′-blocking group. For example, a 3′-blocking group is optionallyremoved and then detected as it is flowed to a waster reservoir in amicrofluidic device.

A detector is placed proximal to a detection region, e.g., proximal tothe immobilized nucleic acid templates and primers, and the labeledcomponents are detected as they bond to the primer. Alternatively, thedetector moves relative to the device to determine the position of alabeled nucleotide, or the like (or, the detector can simultaneouslymonitor a number of spatial positions corresponding to channel regions,e.g., as in a CCD array).

The detector optionally includes or is operably linked to a computer,e.g., which has software for converting detector signal information intosequencing result information, e.g., concentration of a nucleotide,identity f a nucleotide, sequence of the template nucleotide, or thelike. In addition, sample signals are optionally calibrated, e.g., bycalibrating the microfluidic system by monitoring a signal from a knownsource.

A microfluidic system can also employ multiple different detectionsystems for monitoring the output of the system. Detection systems ofthe present invention are used to detect and monitor the materials in aparticular channel region (or other detection region). Once detected,the flow rate and velocity of materials in the channels is alsooptionally measured and controlled.

Particularly preferred detection systems of the present invention areoptical detection systems for detecting an optical property of amaterial within the channels and/or chambers of the microfluidicprovided herein. Such optical detection systems are typically placedadjacent to a microscale channel of a microfluidic device, and are insensory communication with the channel via an optical detection windowthat is disposed across the channel or chamber of the device. Forexample a detector is optionally placed proximal to a particle setcomprising the nucleic acid template and primer of interest, e.g., todetect incorporation of additional nucleotides into the primer.

Optical detection systems include systems that are capable of measuringthe light emitted from material within the channel, the transmissivityor absorbance of the material, as well as the materials' spectralcharacteristics. In preferred aspects, the detector measures an amountof light emitted from the material, such as from a fluorescent orchemiluminescent material, e.g., the labeled products described above.As such, the detection system will typically include collection opticsfor gathering a light based signal transmitted through the detectionwindow, and transmitting that signal to an appropriate light detector.Microscope objectives of varying power, field diameter, and focal lengthare readily utilized as at least a portion of this optical train. Thelight detectors are optionally photodiodes, avalanche photodiodes,photomultiplier tubes, diode arrays, or in some cases, imaging systems,such as charged coupled devices (CCDs) and the like. In preferredaspects, photodiodes are utilized, at least in part, as the lightdetectors. The detection system is typically coupled to a computer(described in greater detail below), via an analog to digital or digitalto analog converter, for transmitting detected light data to thecomputer for analysis, storage and data manipulation.

In the case of fluorescent materials, e.g., labeled nucleotides, thedetector typically includes a light source that produces light at anappropriate wavelength for activating the fluorescent material, as wellas optics for directing the light source through the detection window tothe product contained in the channel or chamber. The light source can beany number of light sources that provides an appropriate wavelength,including lasers, laser diodes and LEDs. Other light sources arerequired for other detection systems. For example, broadband lightsources are typically used in light scattering/transmissivity detectionschemes, and the like. Typically, light selection parameters are wellknown to those of skill in the art.

Similar light sources are also used to provide light of appropriatewavelength for photobleaching as described above. In some embodiments,the same light source is used to detect and photobleach the labelednucleotides that are added to a growing nucleic acid chain.

The detector can exist as a separate unit, but is preferably integratedwith the controller system, into a single instrument. Integration ofthese functions into a single unit facilitates connection of theseinstruments with the computer (described below), by permitting the useof few or a single communication port(s) for transmitting informationbetween the controller, the detector and the computer. For example, thecontroller typically controls the length of a photobleaching pulse.

Computer

As noted above, either or both of the fluid direction system and/or thedetection system are coupled to an appropriately programmed processor orcomputer which functions to instruct the operation of these instrumentsin accordance with preprogrammed or user input instructions, receivedata and information from these instruments, and interpret, manipulateand report this information to the user. As such, the computer istypically appropriately coupled to one or both of these instruments(e.g., including an analog to digital or digital to analog converter asneeded).

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation. For example, the softwareoptionally directs the fluid direction system to transport one or morenucleotides into a main channel, one or more template and primer into amain channel, and any other movement necessary to analyze the results ofthe assay performed.

The computer then receives the data from the one or moresensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as in monitoring and control of flow rates,temperatures, applied voltages, and the like. For example, the voltageson an electrophoretic separation channel are optionally adjusted.

In addition, the computer optionally includes software for deconvolutionof the signal or signals from the detection system. For example, adeconvolution of the data provides concentrations of the nucleotides orintercalating dyes detected

Example Integrated System

FIG. 2, Panels A, B, and C and FIG. 3 provide additional detailsregarding example integrated systems that are optionally used topractice the methods herein. As shown, body structure 202 has mainchannel 210 disposed therein. For example, particle array comprising aset of DNA template and primers is optionally flowed from pipettorchannel 220 towards reservoir 214, e.g., by applying a vacuum atreservoir 214 (or another point in the system) or by applyingappropriate voltage gradients. Reagents, e.g., nucleotides andpolymerase, are optionally flowed into main channel 210 from reservoirs208 and 204 or from pipettor channel 220. The templates incubate in mainchannel 210 with, e.g., polymerase and one or more nucleotides ornucleotide analogs. If an appropriate nucleotide, e.g., complementary tothe template, is present, the polymerase adds the nucleotide to theprimer strand of the nucleic acid, thus extending the double strandedregion of the nucleic acid. A buffer or wash solution is optionallyflowed from reservoir 208, 204, or pipettor channel 220 into mainchannel 210 to inactivate or remove any nucleotides that were notincorporated into the primer strand. Additional materials, such asbuffer solutions, intercalating dyes, other sequencing reagents, and thelike, as described above are optionally flowed into main channel 210.The added nucleotide remains attached to the primer strand and isdetected, e.g., in main channel 210, e.g., at a particle retention areain the downstream end of the channel. Flow from these wells isoptionally performed by modulating fluid pressure, or by electrokineticapproaches as described (or both). The arrangement of channels depictedin FIG. 2 is only one possible arrangement out of many which areappropriate and available for use in the present invention.

Samples and materials are optionally flowed from the enumerated wells orfrom a source external to the body structure. As depicted, theintegrated system optionally includes pipettor channel 220, e.g.,protruding from body 202, for accessing a source of materials externalto the microfluidic system. Typically, the external source is amicrotiter dish or other convenient storage medium. For example, asdepicted in FIG. 3, pipettor channel 220 can access microwell plate 308,which includes sample materials, nucleotides, templates, primers,polymerase, particle arrays, intercalating dyes, and the like, in thewells of the plate.

Detector 310 is in sensory communication with channel 204, detectingsignals resulting, e.g., from labeled nucleotides or nucleic acids.Detector 310 is optionally coupled to any of the channels or regions ofthe device where detection is desired. Detector 310 is operably linkedto computer 304, which digitizes, stores, and manipulates signalinformation detected by detector 310, e.g., using any of theinstructions described above, e.g., or any other instruction set, e.g.,for determining concentration or identity.

Fluid direction system 302 controls voltage, pressure, or both, e.g., atthe wells of the systems or through the channels of the system, or atvacuum couplings fluidly coupled to channel 210 or other channeldescribed above. Optionally, as depicted, computer 304 controls fluiddirection system 302. In one set of embodiments, computer 304 usessignal information to select further parameters for the microfluidicsystem. For example, upon detecting the addition or lack of addition ofa nucleotide to a primer, the computer optionally directs addition of adifferent nucleotide into the system.

Kits

Generally, the microfluidic devices described herein are optionallypackaged to include reagents for performing the device's preferredfunction. For example, the kits optionally include any of microfluidicdevices described along with assay components, reagents, samplematerials, particle sets, control materials, or the like. For example akit for sequencing by synthesis with detection by intercalationtypically includes an intercalating dye and a series of nucleotides,e.g., CTP, GTP, ATP, and TTP. A kit or sequencing by incorporation using3′-blocking groups typically includes a series of nucleotide analogs,such as those of formulas (I) and (II) along with reagents for removingthe 3′-blocking group, such s reducing agents and phosphatases. Suchkits also typically include appropriate instructions for using thedevices and reagents, and in cases where reagents are not predisposed inthe devices themselves, with appropriate instructions for introducingthe reagents into the channels and/or chambers of the device. In thislatter case, these kits optionally include special ancillary devices forintroducing materials into the microfluidic systems, e.g., appropriatelyconfigured syringes/pumps, or the like (in one preferred embodiment, thedevice itself comprises a pipettor element, such as an electropipettorfor introducing material into channels and chambers within the device).In the former case, such kits typically include a microfluidic devicewith necessary reagents predisposed in the channels/chambers of thedevice. Generally, such reagents are provided in a stabilized form, soas to prevent degradation or other loss during prolonged storage, e.g.,from leakage. A number of stabilizing processes are widely used forreagents that are to be stored, such as the inclusion of chemicalstabilizers (i.e., enzymatic inhibitors, microcides/bacteriostats,anticoagulants), the physical stabilization of the material, e.g.,through immobilization on a solid support, entrapment in a matrix (i.e.,a gel), lyophilization, or the like.

Kits also optionally include packaging materials or containers forholding microfluidic device, system or reagent elements.

The discussion above is generally applicable to the aspects andembodiments of the invention described in the claims.

Moreover, modifications can be made to the method and apparatusdescribed herein without departing from the spirit and scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses including the following:

The use of a microfluidic system for sequencing a nucleic acid as setforth herein.

The use of a microfluidic system for sequencing by synthesis orincorporation as set forth herein.

The use of a microfluidic system for sequencing by photobleaching as setforth herein.

The use of a microfluidic system for sequencing by synthesis withdetection of intercalating dyes as described herein.

A sequencing reaction utilizing any of the devices, methods, ornucleotide analogs described herein.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were individually so denoted.

1. A method of sequencing a nucleic acid, the method comprising: (i)hybridizing a nucleic acid template and a primer, producing a hybridizednucleic acid comprising a double-stranded region; (ii) incubating thehybridized nucleic acid with a polymerase and a series of nucleotides,thereby adding at least one nucleotide to the primer, resulting in anextended double-stranded region, wherein the incubating is performed inthe presence of an intercalating dye, which intercalating dyeintercalates into the extended double stranded region; (iii) detectingthe intercalating dye, thereby detecting the addition of the at leastone nucleotide to the primer; and, (iv) repeating steps (ii) through(iv) thereby sequencing the nucleic acid.
 2. The method of claim 1,wherein the nucleic acid is DNA or RNA.
 3. The method of claim 1,wherein the series of nucleotides comprises one or more of:deoxyadenosine 5′-triphosphate, deoxyguanosine 5′-triphosphate,deoxycytidine 5′-triphosphate, deoxythymidine 5′-triphosphate,deoxyuridine 5′-triphosphate, adenosine 5′-triphosphate, guanosine5′-triphosphate, cytidine 5′-triphosphate, uridine 5′-triphosphate, andan analog thereof.
 4. The method of claim 1, wherein the intercalatingdye comprises ethidium, ethidium bromide, an acridine dye, anintercalating nucleic acid stain, a cyanine dye, proflavin, propidiumiodide, acriflavin, proflavin, actinomycin, anthracyclines, ornogalamycin.
 5. The method of claim 1, wherein the intercalating dyeintercalates into the double stranded region and into the extendeddouble-stranded region.
 6. The method of claim 1, wherein the detectingstep comprises detecting a signal difference between the double strandedregion and the extended double stranded region.
 7. The method of claim1, wherein the detecting step further comprises photobleaching theintercalating dye after detecting the intercalating dye or approximatelyconcurrent with detecting the intercalating dye.
 8. The method of claim1, comprising performing the method in a microscale channel.
 9. Themethod of claim 8, wherein the incubating step comprises: (a) incubatingthe nucleic acid double stranded region with a first nucleotide; (b)washing unincorporated nucleotides from the microscale channel anddetecting the intercalating dye; (c) repeating steps (a) and (b) for asecond nucleotide, a third nucleotide, and a fourth nucleotide.
 10. Themethod of claim 9, wherein the first nucleotide, the second, nucleotide,the third nucleotide and the fourth nucleotide each comprise a differentnucleotide.
 11. The method of claim 10, wherein the different nucleotideis selected from: deoxyadenosine 5′-triphosphate, deoxyguanosine5′-triphosphate, deoxycytidine 5′-triphosphate, deoxythymidine5′-triphosphate, deoxyuridine 5′-triphosphate, adenosine5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-triphosphate,uridine 5′-triphosphate, and an analog thereof.
 12. The method of claim1, comprising providing a set of particles, which set of particlescomprises one or more of: the nucleic acid template and the primer. 13.The method of claim 12, wherein the set of particles comprises anordered array.
 14. The method of claim 12, wherein the set of particlescomprises about 1 or more particles, about 10 or more particles, about100 or more particles, about 1000 or more particles, or about 10,000 ormore particles.
 15. The method of claim 12, wherein the set of particlescomprises a set of beads, which beads are selected from: polymer beads,silica beads, ceramic beads, clay beads, glass beads, magnetic beads,metallic beads, paramagnetic beads, inorganic beads, and organic beads;and wherein the beads have a shape, which shape is selected from one ormore of: spherical, helical, cylindrical, spheroid, irregular, rodshaped, cone shaped, cubic, and polyhedral.
 16. The method of claim 12,comprising positioning the set of particles within a microscale channel.17. The method of claim 12, wherein the incubating step comprisesflowing the polymerase across the set of particles or flowing the set ofparticles through the polymerase.
 18. A method of sequencing a nucleicacid, comprising: (i) providing a first set of particles comprising atleast one set of nucleic acid templates in a first microfluidic channel;(ii) flowing a train of reagents comprising a plurality of sequencingreagents across the first set of particles, or flowing the first set ofparticles through the reagent train, thereby sequentially contacting theat least one set of nucleic acid templates with the plurality ofsequencing reagents; (iii) detecting a signal resulting from exposure ofthe first set of particles to the reagent train, thereby providing aportion of sequence of the nucleic acid template; and, repeating steps(ii) and (iii), thereby sequencing the nucleic acid.
 19. The method ofclaim 18, wherein the nucleic acid templates comprise DNA or RNA. 20.The method of claim 18, wherein the train of reagents comprises one ormore of: a template, a primer, a polymerase, a sufurylase, an apyrase,an inorganic phosphate, ATP, a thermostable polymerase, luciferin,luciferase, an endonuclease, an exonuclease, Mg⁺⁺, a molecular crowdingagent, a buffer, a dNTP, a salt, a phosphatase, a reducing agent, amodified dNTP, a nucleotide, a nucleotide analog, a nucleotide analogcomprising a 3′-blocking group, a nucleotide analog comprising a3′-phosphate group, a nucleotide analog comprising a 3′-carbamate group,a chain-terminating nucleotide analog, a reversible chain terminatingnucleotide analog, a fluorescently labeled nucleotide, and anintercalating dye.
 21. The method of claim 18, wherein the plurality ofsequencing reagents comprises at least one of: a compound having formula(I), and a compound having formula (II):

wherein R⁴ comprises one or more of a linker moiety and a detectablelabel and B comprises one or more of a nitrogenous base and thedetectable label.
 22. The method of claim 21, wherein the linker moietycomprises an acyl, an S-acyl, an alkyl, an aromatic, or anheteroaromatic group.
 23. The method of claim 21, wherein thenitrogenous base comprises adenine, guanine, thymine, cytosine, oruracil.
 24. The method of claim 18, wherein step (ii) comprises: (a)flowing at least one set of nucleic acid primers across the set ofparticles or flowing the set of particles through at least one set ofnucleic acid primers, thereby contacting the nucleic acid templates withthe nucleic acid primers and forming a double-stranded region; and, (b)flowing a polymerase and one or more nucleotides or nucleotide analogsacross the set of particles or flowing the set of particles through thepolymerase and the one or more nucleotides or nucleotide analogs,thereby adding at least one of the one or more nucleotides or nucleotideanalogs to at least one of the nucleic acid primers, resulting in anextended double-stranded region.
 25. The method of claim 24, wherein theone or more nucleotide analogs comprise chain-terminating nucleotideanalogs.
 26. The method of claim 24, wherein the chain-terminatingnucleotide analogs comprise reversible chain-terminating nucleotideanalogs.
 27. The method of claim 24, wherein the one or more nucleotideanalogs comprise a 3′-blocking group.
 28. The method of claim 27,wherein the 3′-blocking group comprises a carbamate group or a phosphategroup.
 29. The method of claim 27, the method further comprisingremoving the 3′-blocking group from the extended double-stranded region,resulting in an extendable double-stranded region.
 30. The method ofclaim 29, wherein the removing the 3′-blocking group comprises flowing areducing agent and a phosphatase across the set of particles or flowingthe set of particles through the reducing agent and the phosphatase. 31.The method of claim 30, wherein the reducing agent is selected fromdiborane, dithiothreitol, glutathione, TCEP, and disulfide reductase.32. The method of claim 24, wherein the one or more nucleotides comprisea series of nucleoside 5′-triphosphates and the method further comprisesflowing an intercalating dye across the set of particles or flowing theset of particles through an intercalating dye.
 33. The method of claim32, wherein detecting the signal comprises detecting the intercalatingdye.
 34. The method of claim 32, the method further comprisingphotobleaching the intercalating dye after detecting the intercalatingdye or concurrent with detecting the intercalating dye.
 35. The methodof claim 18, wherein step (iii) comprises photobleaching the at leastone of the one or more nucleotides or nucleotide analogs.
 36. The methodof claim 18, wherein one or more of: the first set of particles and thereagent train, is flowed through the microscale channel by one or moreof: pressure, centripetal force, centrifugal force, a moving magneticfield, and an electrokinetic force.
 37. The method of claim 18, whereinthe first set of particles comprises an ordered array.
 38. The method ofclaim 18, wherein the first set of particles comprises about 1 or moreparticles, about 10 or more particles, about 100 or more particles,about 1000 or more particles, or about 10,000 or more particles.
 39. Themethod of claim 18, wherein the first set of particles comprises a setof beads, which beads are selected from: polymer beads, silica beads,ceramic beads, clay beads, glass beads, magnetic beads, metallic beads,paramagnetic beads, inorganic beads, and organic beads; and wherein thebeads have a shape, which shape is selected from one or more of:spherical, helical, cylindrical, spheroid, irregular, rod-shaped,cone-shaped, cubic, and polyhedral.
 40. The method of claim 18, whereinthe train of reagents comprises reagents for sequencing the nucleic acidtemplates by pyrosequencing, by incorporation, by synthesis, byphotobleaching, or by intercalation.
 41. The method of claim 18, furthercomprising reiteratively repeating steps (ii) and (iii) until at leastone reagent in the reagent train becomes depleted, the method furthercomprising flowing a second train of reagents comprising a plurality ofsequencing reagents across the first set of particles, or flowing thefirst set of particles through the second reagent train.
 42. The methodof claim 18, wherein the first set of particles is present in an arrayof particle sets, the method further comprising flowing the train ofreagents across the array of particle sets, or flowing the array ofparticle sets through the reagent train.